Purified and recombinant antigenic protein associated with abdominal aortic aneurysm (AAA) disease, and diagnostic and therapeutic use thereof

ABSTRACT

This invention provides an isolated protein of approximately 40 kDa which is purified from human aortic tissue and immunoreactive with AAA-associated immunoglobulin. Also provided are a method of diagnosing AAA disease in a subject using said isolated protein and a pharmaceutical composition comprising said isolated protein. A method of alleviating AAA disease in a subject comprising administering said pharmaceutical composition comprising the isolated protein is also provided. The subject invention also provides a recombinantly produced human aortic protein which is immunoreactive with AAA-associated immunoglobulin. Also provided are a method of diagnosing AAA disease in a subject using said recombinantly produced protein and a pharmaceutical composition comprising said recombinantly produced protein. A method of alleviating AAA disease in a subject comprising administering said pharmaceutical composition comprising the recombinantly produced protein is also provided.

This application is a divisional of U.S. Ser. No. 08/812,586, filed Mar.7, 1997, now U.S. Pat. No. 6,048,704, which claims priority of U.S.Provisional Application Serial No. 60/012,976, filed Mar. 7, 1996, thecontents of which are hereby incorporated by reference into the presentapplication.

Throughout this application, various references are referred to inparentheses. Disclosures of these publication in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the claims.

BACKGROUND OF THE INVENTION

Microfibrils, 10-14 nm in diameter, are extracellular matrix proteinswhich play important roles in the assembly and maintenance of elasticfibers.^(1.2) A bovine microfibril-associated glycoprotein (MAGP) withMW 31 kDa was discovered in 1986,^(3,4) and then cloned in 1994.⁵Kobayashi et al. Reported a bovine MAGP-36 with calcium-binding andfibrinogen-like domains, with a tissue distribution uniquely limited toaorta.⁶ A human MAGP, implicated to Smith Magenis syndrome, wassequenced in 1995.⁷

Immunoglobulin (IgG) purified from the aortic wall of patients withabdominal aortic aneurysms (AAAs) is immunoreactive with a human aorticprotein that is homologous to MAGP-36.8.⁸ As described herein, theprotein has been purified and partially sequenced. It has vitronectinand fibrinogen-like domains, along with a putative calcium-bindingdomain.⁹ It is designated Aortic Aneurysm-Autoantigenic Protein—40 kDaor Aortic-Aneurysm-Associated Protein-40 (AAAP-40).⁹

Another matrix protein detected in human embryonic tissue (sulfatedprotein 30 kDa=SP-30) has been reported to be immunoreactive withmonoclonal antibodies against human vitronectin, and it co-distributesin tissue with the Protein that is immunoreactive with antibody againstMAGP-31. A sequence in AAAP-40 matches residues #230-240 in humanvitronectin. It was demonstrated that AAAP-40 was immunoreactive withrabbit anti-human vitronectin antibody. In addition, the experimentsdescribed in this application were carried out to clone the cDNAencoding AAAP-40, express it as a recombinant, and assign itconclusively as a human MAGP.

Nomenclature of the microfibrillar proteins associated with the elastinfiber is confusing. A principal component of the microfibril isfibrillin (fib-15), discovered by Sakai, et al.,¹ and Marfan's syndromehas been traced to mutations in the gene for fibrillin on chromosome15.² ³ ⁴ A bovine microfibrillar protein (Mr apx. 31 kDa) was discoveredin 1986 by Gibson, et al.,⁵ ⁶ who coined the term“microfibril-associated glycoprotein”(MAGP). Bashir, et al. have alsocloned the gene for this protein.⁷ Kobayashi, et al. reported a 36 kDacalcium-binding protein, also in cow, with tissue distribution uniquelylimited to the aorta (MAGP-36).⁸ The second human MAGP (deduced MW 21kDa) was recently reported to have an open reading frame of 255 aminoacids and to be linked to Smith Magenis syndrome.⁹ The authors of thepaper describing the Smith Magenis protein prefer the abbreviation“MFAP”, to avoid confusion with abbreviations for microfilamentousproteins.

It has been reported that IgG from the aortic wall of patients withabdominal aortic aneurysms (AAA) is immunoreactive with a human aorticprotein (MW apx. 80 kDa) that has features of the bovine aortic proteinof Kobayshi, et al. (MAGP-36).¹⁰ MAGP-36 occurs in nature as adisulfide-bonded dimer, so further tissue extractions under reducingconditions as described by Prosser, et al. were carried out as describedherein.¹¹ This approach has led to the partial characterization of aprotein of apx. 40 kDa that is immunoreactive with AAA IgG. This proteinis called Aortic Aneurysm-Autoantigenic Protein—40 kDa orAortic-Aneurysm-Associated Protein-40 (AAAP-40). The present applicationdescribes its partial sequence and suggests that, since it is the thirdhuman microfibrillar protein to be described, it be called MAGP-3.

SUMMARY OF THE INVENTION

This invention provides an isolated protein of approximately 40 kDawhich is purified from human aortic tissue and immunoreactive withAAA-associated immunoglobulin. This invention provides an isolatedprotein has the amino acid sequence set forth in SEQ ID NO:1.

This invention provides a recombinantly produced human aortic proteinwhich is immunoreactive with both AAA-associated immunoglobulin andhuman kappa immunoglobulin and having a molecular weight ofapproximately 24 kDa-28 kDa.

This invention further provides an isolated nucleic acid moleculeencoding a human aortic protein. In an embodiment, the isolated nucleicacid, cDNA, designated AAAP-CL1, encodes a human aortic protein which isimmunoreactive with both AAA-associated immunoglobulin and human kappaimmunoglobulin and has a molecular weight of approximately 24 kDa-28kDa. In another embodiment, the isolated cDNA, designated AAAP-CL5,encodes a human aortic protein which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin and has amolecular weight of approximately 24 kDa-28 kDa. In yet anotherembodiment, the isolated cDNA, designated AAAP-CL4, encodes a humanaortic protein which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin and has a molecular weightof approximately 24 kDa-28 kDa.

This invention provides a recombinantly produced human aortic proteinwhich is immunoreactive with AAA-associated immunoglobulin. In anembodiment, the protein is of a molecular weight of approximately 40kDa.

This invention provides a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:2 which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin.

This invention provides a purified recombinant human aortic protein ofapproximately 28 kDa having the amino acid sequence set forth in SEQ IDNO:3 which is immunoreactive with both AAA-associated immunoglobulin andhuman kappa immunoglobulin.

This invention provides a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:4 which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin.

This invention provides an antibody directed to a purified humanprotein. This invention further provides an antibody capable ofspecifically recognizing human aortic protein.

This invention provides a method of diagnosing AAA disease in a subjectwhich comprises: (a) obtaining a suitable sample from the subject; (b)contacting the sample with the protein of either the isolated protein ofapproximately 40 kDa which is purified from human aortic tissue andimmunoreactive with AAA-associated immunoglobulin or the isolatedprotein having the amino acid sequence set forth in SEQ ID NO:1 underconditions permitting the protein to bind to AAA-associatedimmunoglobulin if present in the sample; and (c)determining the presenceof bound protein, the presence of bound protein being indicative of AAAdisease, thereby diagnosing AAA disease in the subject.

This invention provides a pharmaceutical composition for alleviating AAAdisease in a subject which comprises the protein of approximately 40 kDawhich is purified from human aortic tissue and immunoreactive withAAA-associated immunoglobulin and a pharmaceutically acceptable carrier.

This invention provides a method of alleviating AAA disease in a subjectwhich comprises administering to the subject an amount of theaforementioned composition effective to induce tolerance to antigenicAAA protein in the subject.

This invention provides a method of diagnosing AAA disease in a subjectwhich comprises: (a) obtaining a suitable sample from the subject; (b)contacting the sample with the protein selected from any of thefollowing proteins: the isolated protein of approximately 40 kDa whichis purified from human aortic tissue and immunoreactive withAAA-associated immunoglobulin; the isolated protein of approximately 40kDa which is purified from human aortic tissue and immunoreactive withAAA-associated immunoglobulin having the amino acid sequence set forthin SEQ ID NO:1; a recombinantly produced human aortic protein which isimmunoreactive with AAA-associated immunoglobulin; a recombinantlyproduced human aortic protein which is immunoreactive withAAA-associated immunoglobulin and is of a molecular weight ofapproximately 40 kDa; a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:2 which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin; a purified recombinanthuman aortic protein of approximately 28 kDa having the amino acidsequence set forth in SEQ ID NO:3 which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin; a purifiedrecombinant human aortic protein of approximately 24 kDa-28 kDa havingthe amino acid sequence set forth in SEQ ID NO:4 which is immunoreactivewith both AAA-associated immunoglobulin and human kappa immunoglobulinunder conditions permitting the protein to bind to AAA-associatedimmunoglobulin if present in the sample; and (c) determining thepresence of bound protein, the presence of bound protein beingindicative of AAA disease, thereby diagnosing AAA disease in thesubject. In an embodiment, the subject may be a mammal, for example ahuman subject.

This invention provides a pharmaceutical composition for alleviating AAAdisease in a subject which comprises the protein is selected from any ofthe following proteins: the isolated protein of approximately 40 kDawhich is purified from human aortic tissue and immunoreactive withAAA-associated immuinoglobulin; the isolated protein of approximately 40kDa which is purified from human aortic tissue and immunoreactive withAAA-associated immunoglobulin having the amino acid sequence set forthin SEQ ID NO:1; a recombinantly produced human aortic protein which isimmunoreactive with AAA-associated immunoglobulin; a recombinantlyproduced human aortic protein which is immunoreactive withAAA-associated immunoglobulin and is of a molecular weight ofapproximately 40 kDa; a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:2 which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin; a purified recombinanthuman aortic protein of approximately 28 kDa having the amino acidsequence set forth in SEQ ID NO:3 which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin; a purifiedrecombinant human aortic protein of approximately 24 kDa-28 kDa havingthe amino acid sequence set forth in SEQ ID NO:4 which is immunoreactivewith both AAA-associated immunoglobulin and human kappa immunoglobulinand a pharmaceutically acceptable carrier.

This invention provides a kit for detecting the presence ofAAA-associated immunoglobulin in a sample, which comprises: (a) a solidsupport having a plurality of covalently linked probes which may be thesame or different, each probe of which comprises a human aortic proteinwhich is capable of binding AAA-associated immunoglobulin; and (b) ameans for determining the presence of AAA-associated immunoglobulinbound to the human aortic protein. In an embodiment of the kit, thehuman aortic protein is selected from any of the following proteins: theisolated protein of approximately 40 kDa which is purified from humanaortic tissue and immunoreactive with AAA-associated immunoglobulin; theisolated protein of approximately 40 kDa which is purified from humanaortic tissue and immunoreactive with AAA-associated immunoglobulinhaving the amino acid sequence set forth in SEQ ID NO:1; a recombinantlyproduced human aortic protein which is immunoreactive withAAA-associated immunoglobulin; a recombinantly produced human aorticprotein which is immunoreactive with AAA-associated immunoglobulin andis of a molecular weight of approximately 40 kDa; a purified recombinanthuman aortic protein of approximately 24 kDa-28 kDa having the aminoacid sequence set forth in SEQ ID NO:2 which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin; a purifiedrecombinant human aortic protein of approximately 28 kDa having theamino acid sequence set forth in SEQ ID NO:3 which is immunoreactivewith both AAA-associated immunoglobulin and human kappa immunoglobulin;a purified recombinant human aortic protein of approximately 24 kDa-28kDa having the amino acid sequence set forth in SEQ ID NO:4 which isimmunoreactive with both AAA-associated immunoglobulin and human kappaimmunoglobulin.

This invention provides a kit for detecting the presence of an elevatedor abnormal level of human aortic protein in a sample, whichcomprises:(a) a plurality of covalently linked probes which may be thesame or different, each probe of which comprises any one of aAAA-associated immunoglobulin and a human kappa immunoglobulin which iscapable of binding human aortic protein; and (b) a means for determiningthe elevated or abnormal level of human aortic protein bound toAAA-associated immunoglobulin or human kappa immunoglcbulin bycomparison to a normal level of human aortic protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.

Immunoreactive proteins are located surrounding the aortic adventitialelastic fibers in both AAA tissue and normal aortic tissue sections whenincubated with IgG from AAA wall or serum. Tissue sections incubatedwith second antibody alone showed no immunoreactivity.

AAA: abdominal aortic aneurysm tissue section. NL: normal abdominalaortic tissue section. EVG: elastin Von-Gleson stain.

FIG. 2.

Soluble AAA extracts were separated by SDS/PAGE, and then probed firstwith either IgG from AAA wall or serum, and then with APC rabbitanti-human IgG. Unique immunoreactive binding was observed at 40 kDa(arrow), suggesting the presence of AAAP-40. Second antibody alone wasused as control, demonstrating IgGs in the soluble extracts of theaorta.

FIG. 3.

Computer-enhanced Western immunoblot. Soluble AAA extracts wereseparated by SDS/PAGE, and then probed first with rabbit anti-humanvitronectin antibody, and then with APC goat anti-rabbit IgG. Uniqueimmunoreactive material was observed at MW 40 (arrow). Peripherallymphocytes were harvested, separated by SDS/PAGE, and probed in thesame manner. Immunoreactive proteins at MW 75 and 65 kDa, consistentwith lymphocyte-bound vitronectin and its degraded form, ware alsoobserved in the aortic extracts.

STD: Standard molecular marker. AAA:AAA PBS tissue extracts. LNC:Lymphocytes.

FIG. 4.

Computer-enhanced glycoprotein detection blot. Soluble AAA extracts andcontrol glycoproteins were separated by SDS/PAGE. Assays for galanthusnivalis agglutinin (GNA), sambucus nigra agglutinin (SNA), maackiaamurensis agglutinin (MAA), datura stramonium agglutinin (DSA), andpeanut agglutinin (PNA) were undertaken. Control glycoproteincarboxypeptidase Y (CBPXY) reacted with GNA, indicating terminallylinked mannose. Control glycoprotein transferrin (TRNSF) reacted withSNA, indicating sialic acid terminally linked alpha (2-6) to galactoseor N-acetylgalactosamine. Control glycoprotein fetuin (FETN) reactedwith SNA, MAA (indicating sialic acid terminally linked alpha (2-3) togalactose), and DSA (indicating galactose beta (1-4) galactose-beta(1-4)-N-acetylglucosamine). Control glycoprotein asialofetuin (ASFTN)reacted with DSA, and PNA, indicating galactose-beta(1-3)-N-acetylgalactosamine. Several reactive bands were observed in thelanes of AAA extracts, demonstrating the presence of solubleglycoproteins. AAA tissue extracts tested with MAA, DSA and PNAdemonstrated positive immunoreactivity at MW 40 kDa. These resultsindicate that AAAP-40 has carbohydrate moieties including; sialic acidterminally linked alpha (2-3) to galactose, galactose beta(1-3)-N-acetylgalactosamine, and galactose beta(1-4)-N-acetylgalactosamine.

FIG. 5.

Photomicrographs of immunmohistochemical preparations of aneurysmal(AAA) and normal (NL) aortic advencitia, illustrating binding of rabbitanti-human Ig kappa to the elastin-associated microfibril. Controlexperiments with rabbit anti-human Ig heavy chain as first antibodyrevealed minimal, if any, immunoreactivity of the micro-fibril.

FIG. 6.

Transformed cells and control cells (XL) were incubated with IPTG toinduce protein expression. After the cells were lysed, the solubleproteins were separated by SDS/PAGE (12.5%) and probed with purifiedIgGs from AAA patients. AAA serum IgG was uniquely immunoreactive withrAAAP-CL1 and rAAAP-CL5 at approximately 28 kDa, not present in apreparation of the normal bacterial cells.

FIG. 7.

Transformed cells and control cells (XL) were incubated with IPTG toinduce protein expression. After the cells were lysed, the solubleproteins were separated by SDS/PAGE (12.5%) and probed with purifiedserum IgGs from normal patients. No unique immunoreactive material wasdetected.

FIG. 8.

Transformed cells and control cells (XL) were incubated with IPTG toinduce protein expression. After the cells were lysed, the solubleproteins were separated by SDS/PAGE (12.5%) and probed with rabbitanti-human Ig kappa antibody. Unique immunoreactive binding was detectedwith rAAAP-CL1 and rAAAP-CL5 at approximately 24 kDa, not present in apreparation of the normal bacterial cells.

FIG. 9.

Transformed cells and control cells (XL) were incubated with IPTG toinduce protein expression. After the cells were lysed, the solubleproteins were separated by SDS/PAGE (12.5%) and probed with APC goatanti-human heavy chain antibody. No unique immunoreactive binding wasdetected.

FIGS. 10A-F.

(A) rAAAP-CL1 as modeled by SwissModel, labeled in the standardconvention for Ig-folds [¹⁹] and displayed by RasMol in “cartoon” modewith yellow coding for beta strands and red coding for alpha coils.There is a short helix between strands e and f.

(B) The molecule, rAAAP-CL1, displayed by RasMol in “group” mode,wherein N-terminal progresses to C-terminal in colors from blue>darkgreen>light green>yellow>red.

(C) rAAAP-CL1 rotated horizontally 180 degrees to display the back ofthe bottom beta sheet, in “group” mode.

(D) The first Ig-like domain of VCAM-1 mouse. The c, f, and g strands ofthe bottom sheet are not present, but the structure has substantialsimilarity to the front sheet of rAAAP-CL1, including the alpha helix atthe end of strand e. This degree of three dimensional structuralsimilarity is interesting, considering that less than 30% of the aminoacid residues have been converted (as shown in E)

(E) A Pairwise comparison of rAAAP-CL1 and VCAM-1 mouse displayed byLALNView.

(F) A Pairwise comparison of rAAAP-CL1 with protein from cytomegalovirus

FIG. 11.

A hypothetical evolutionary tree based on PAM distances calculated byAllAll.

FIG. 12.

Transformed cells (rAAA) and control cells (XL) were incubated with ITPGto induce protein expression. After the cells were lysed, the solubleproteins were separated by SDS/PAGE (12.5%) and probed with purifiedIgGs from either AAA patients or volunteers. AAA IgG was immunoreactivewith a protein at 28 kDa, which was not present in a preparation of thenormal bacterial cells.

FIG. 13.

Pair-wise alignment of the amino acid sequence of rAAAP-CL4 (clone 4)with human fibrinogen-beta, microfibril associated protein-4 (MFAP-4)((26) in 4th series), and microfibril-associated glycoprotein-36(MAGP-36) ((4) in 4th series).

FIG. 14.

Pair-wise alignment of rAAAP-CL4 (clone 4) with two immunoglobulin kappasequences that we have reported to resemble clones 1 and 5.((7) in 4thseries)

FIG. 15.

Pair-wise alignment of rAAAP-CL4 (clone 4) with a protein fromcytomegalovirus ((28) in 4th series) and a protein from influenza ((30)in 4th series).

FIG. 16.

Swiss-Model of the three-dimensional structure of MAT-CAM 2 revealing itto have an immunoglobulin-like sandwich, formed by two antiparallel betasheets, connected by a helix in the region of the putative binding site.

FIG. 17.

Pair-wise comparison of MAT-CAMs 1 (Clone 1) and 2 (Clone 2), asdisplayed by LalnView, with color coding for the regions of greatestsimilarity in amino acid seqaence. The three dimensional structures havebeen conserved despite substantial divergence in sequence.

FIG. 18.

An evolutionary tree, roughly scaled in PAM units (point acceptedmutations/100 amino acid residues), as computed by AllAll. For proteinsof the fibrinogen family, a PAM unit has been estimated to be about 1.1million years.¹⁴

FIG. 19.

Histological section of aneurysmal aorta probed with antibodies againstimmunoglobulin kappa and against immunoglobulin heavy chain. Anti-Igkappa localizes to the adventitial elastin-associated fibril.

FIG. 20.

In-situ hybridization of clone 1 against a histological section ofnormal human aorta. The predominant cell type positive for hybridizationin this section is mesenchymal, although further studies would berequired to differentiate whether the positive cells are fibroblasts orsmooth muscle cells.

FIG. 21.

Tissue samples of AAA and control aortas were sectioned at 6 μm forhistochemistry (iron hematoxylin, abbreviated EVG) andimmunohistochemistry with rabbit anti-T pallidum (Tpall) and rabbitanti-herpes simplex virus (HSV). The EVG stain shows fibers of elastinin the aortic adventitia. No immunoreactivity is seen with secondantibody alone, although the elastin fibers can be faintly recognized bytheir optical property of birefringence. The anti-T pall and anti-herpesantibodies are immunoreactive with the periphery of the elastic fibers,consistent with the elastin-associated microfibril. (Light microscopy,400x.).

FIG. 22.

Treponema pallidum (Tpall) proteins were separated by SDS/PAGE,transferred to membrane, and then reacted with the following firstantibodies: rabbit anti-T pall, rabbit anti-herpes simplex (HSV), IgGfrom a healthy control, and IgG from the wall of an AAA. Second antibodyalone (anti-rabbit) was used in a control experiment, and it did notbind to the T pall proteins. Rabbit anti-T pall antibody demonstratedmany immunoreactive proteins as expected, ranging in molecular weightfrom 19 kDa to 101 kDa. Anti-herpes simplex antibody was alsoimmunoreactive with several T pall proteins, ranging from 38 to 80 kDa.Second antibody alone for the human IgG experiments was notimmunoreactive. Several faint bands were detectable with control serum,but this weak immunoreactivity may be nonspecific or due to otherepitopes that T pall may share with other pathogens. AAA wall IgG wasconspicuously immunoreactive with several T pall proteins, particularlythe one at approximately 40 kDa.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides an isolated protein of approximately 40 kDawhich is purified from human aortic tissue and immunoreactive withAAA-associated immunoglobulin. In one embodiment, the isolated proteinis capable of forming a disulfide-bonded dimer of approximately 80 kDa.In another embodiment, purification of the protein from human aortictissue comprises extraction of the protein under reducing conditions. Instill another embodiment, the isolated protein has the amino acidsequence set forth in SEQ ID NO:1.

As used herein, AAA means “Abdominal Aortic Aneurysm”.

This invention provides a recombinantly produced human aortic proteinwhich is immunoreactive with both AAA-associated immunoglobulin andhuman kappa immunoglobulin and having a molecular weight ofapproximately 24 kDa-28 kDa.

This invention provides an isolated nucleic acid molecule encoding ahuman aortic protein. In one embodiment, the isolated nucleic acidmolecule is a DNA molecule. In another embodiment, the isolated DNAmolecule is a cDNA molecule. In an embodiment, the isolated cDNA,designated AAAP-CL1, encodes a human aortic protein which isimmunoreactive with both AAA-associated immunoglobulin and human kappaimmunoglobulin and has a molecular weight of approximately 24 kDa-28kDa. In another embodiment, the isolated cDNA, designated AAAP-CL5,encodes a human aortic protein which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin and has amolecular weight of approximately 24 kDa-28 kDa. In yet anotherembodiment, the isolated cDNA, designated AAAP-CL4, encodes a humanaortic protein which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin and has a molecular weightof approximately 24 kDa-28 kDa. In an embodiment, the isolated DNAmolecule is genomic DNA molecule. In one embodiment, the isolatednucleic acid molecule is an RNA molecule. In an embodiment, the isolatednucleic acid molecule encodes a human aortic protein. This inventionprovides a recombinantly produced human aortic protein which isimmunoreactive with AAA-associated immunoglobulin. In an embodiment, theprotein is of a molecular weight of approximately 40 kDa.

This inveition provides a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:2 which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin.

This invention provides a purified recombinant human aortic protein ofapproximately 28 kDa having the amino acid sequence set forth in SEQ IDNO:3 which is immunoreactive with both AAA-associated immunoglobulin andhuman kappa immunoglobulin.

This invention provides a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:4 which is immunoreactive with both AAA-,associatedimmunoglobulin and human kappa immunoglobulin.

This invention provides an antibody directed to a purified humanprotein, said protein selected from the isolated protein ofapproximately 40 kDa which is purified from human aortic tissue andimmunoreactive with AAA-associated immunoglobulin; the isolated proteinof approximately 40 kDa which is purified from human aortic tissue andimmunoreactive with AAA-associated immunoglobulin having the amino acidsequence set forth in SEQ ID NO:1; a recombinantly produced human aorticprotein which is immunoreactive with AAA-associated immunoglobulin; arecombinantly produced human aortic protein which is immunoreactive withAAA-associated immunoglobulin and is of a molecular weight ofapproximately 40 kDa; a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:2 which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin; a purified recombinanthuman aortic protein of approximately 28 kDa having the amino acidsequence set forth in SEQ ID NO:3 which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin; a purifiedrecombinant human aortic protein of approximately 24 kDa-28 kDa havingthe amino acid sequence set forth in SEQ ID NO:4 which is immunoreactivewith both AAA-associated immunoglobulin and human kappa immunoglobulin,other than the AAA-associated immunoglobulin. The aforementionedantibodies may be monoclonal antibodies.

This invention provides an antibody capable of specifically recognizinghuman aortic protein, said protein selected from the isolated protein ofapproximately 40 kDa which is purified from human aortic tissue andimmunoreactive with AAA-associated immunoglobulin; the isolated proteinof approximately 40 kDa which is purified from human aortic tissue andimmunoreactive with AAA-associated immunoglobulin having the amino acidsequence set forth in SEQ ID NO:1; a recombinantly produced human aorticprotein which is immunoreactive with AAA-associated immunoglobulin; arecombinantly produced human aortic protein which is immunoreactive withAAA-associated immunoglobulin and is of a molecular weight ofapproximately 40 kDa; a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:2 which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin; a purified recombinanthuman aortic protein of approximately 28 kDa having the amino acidsequence set forth in SEQ ID NO:3 which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin; a purifiedrecombinant human aortic protein of approximately 24 kDa-28 kDa havingthe amino acid sequence set forth in SEQ ID NO:4 which is immunoreactivewith both AAA-associated immunoglobulin and human kappa immunoglobulin,other than the AAA-associated immunoglobulin. The aforementionedantibodies may be monoclonal antibodies. Methods of making antibodies,including monoclonal antibodies, are well known to one of ordinary skillin the art.

This invention further provides a method of diagnosing AAA disease in asubject which comprises: (a) obtaining a suitable sample from thesubject; (b) contacting the sample with the isolated protein of theinvention under conditions permitting the protein to bind toAAA-associated immunoglobulin if present in the sample; and (c)determining the presence of bound protein, the presence of bound proteinbeing indicative of AAA disease, thereby diagnosing AAA disease in thesubject.

This invention provides a method of diagnosing AAA disease in a subjectwhich comprises:(a) obtaining a suitable sample from the subject; (b)contacting the sample with the protein of either the isolated protein ofapproximately 40 kDa which is purified from human aortic tissue andimmunoreactive with AAA-associated immunoglobulin or the isolatedprotein having the amino acid sequence set forth in SEQ ID NO:1 underconditions emitting the protein to bind to AAA-associated immunoglobulinif present in the sample; and (c) determining the presence of boundprotein, the presence of bound protein being indicative of AAA disease,thereby diagnosing AAA disease in the subject.

This invention provides a method of diagnosing AAA disease in a subjectwhich comprises: (a) obtaining a suitable sample from the subject; (b)contacting the sample with the protein selected from any of thefollowing proteins: the isolated protein of approximately 40 kDa whichis purified from human aortic tissue and immunoreactive withAAA-associated immunoglobulin; the isolated protein of approximately 40kDa which is purified from human aortic tissue and immunoreactive withAAA-associated-immunoglobulin having the amino acid sequence set forthin SEQ ID NO:1; a recombinantly produced human aortic protein which isimmunoreactive with AAA-associated immunoglobulin; a recombinantlyproduced human aortic protein which is immunoreactive withAAA-associated immunoglobulin and is of a molecular weight ofapproximately 40 kDa; a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:2 which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin; a purified recombinanthuman aortic protein of approximately 28 kDa having the amino acidsequence set forth in SEQ ID NO:3 which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin; a purifiedrecombinant human aortic protein of approximately 24 kDa-28 kDa havingthe amino acid sequence set forth in SEQ ID NO:4 which is immunoreactivewith both AAA-associated immunoglobulin and human kappa immunoglobulinunder conditions permitting the protein to bind to AAA-associatedimmunoglobulin if present in the sample; and (c) determining thepresence of bound protein, the presence of bound protein beingindicative of AAA disease, thereby diagnosing AAA disease in thesubject.

Bound protein, i.e. protein bound to AAA-associated immunoglobulin maybe detected using methods well known to those of ordinary skill in theart. Examples of method for detection of bound protein are Enzyme LinkedImmunosorbant Assays (Elisa) and Radioimmunoassays. Assays for detectingbound protein may also be found in texts well known in the art, such asHarlow, E., and Lane, D., 1988.

As used herein, AAA-associated immunoglobulin is immunoglobulin which ispresent in a subject afflicted with abdominal aortic aneurysms (AAA),and which is not present in healthy subjects, i.e. subjects which do notcurrently have AAA.

In the aforementioned methods of the subject invention, the subject maybe a mammal, for example, a human.

The subject invention further provides a pharmaceutical composition foralleviating AAA disease in a subject which comprises the aforementionedpurified protein of the invention and a pharmaceutically acceptablecarrier. This invention provides a pharmaceutical composition foralleviating AAA disease in a subject which comprises the protein ofapproximately 40 kDa which is purified from human aortic tissue andimmunoreactive with AAA-associated immunoglobulin and a pharmaceuticallyacceptable carrier.

This invention provides a pharmaceutical composition for alleviating AAAdisease in a subject which comprises the protein is selected from any ofthe following proteins: the isolated protein of approximately 40 kDawhich is purified from human aortic tissue and immunoreactive withAAA-associated immunoglobulin; the isolated protein of approximately 40kDa which is purified from human aortic tissue and immunoreactive withAAA-associated immunoglobulin having the amino acid sequence set forthin SEQ ID NO:1; a recombinantly produced human aortic protein which isimmunoreactive with AAA-associated immunoglobulin; a recombinantlyproduced human aortic protein which is immunoreactive withAAA-associated immunoglobulin and is of a molecular weight ofapproximately 40 kDa; a purified recombinant human aortic protein ofapproximately 24 kDa-28 kDa having the amino acid sequence set forth inSEQ ID NO:2 which is immunoreactive with both AAA-associatedimmunoglobulin and human kappa immunoglobulin; a purified recombinanthuman aortic protein of approximately 28 kDa having the amino acidsequence set forth in SEQ ID NO:3 which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin; a purifiedrecombinant human aortic protein of approximately 24 kDa-28 kDa havingthe amino acid sequence set forth in SEQ ID NO:4 which is immunoreactivewith both AAA-associated immunoglobulin and human kappa immunoglobulinand a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers. Thepharmaceutical composition may be constituted into any form suitable forthe mode of administration selected. Compositions suitable for oraladministration include solid forms, such as pills, capsules, granules,tablets, and powders, and liquid forms, such as solutions, syrups,elixirs, and suspensions. Forms useful for parenteral administrationinclude sterile solutions, emulsions, and suspensions. In an embodiment,the pharmaceutical composition is suitable for administering orally to asubject.

This invention further provides a method of alleviating AAA disease in asubject which comprises administering to the subject an amount of any ofthe aforementioned compositions comprising the invented purifiedproteins, said amount effective to induce tolerance to antigenic AAAprotein in the subject. In the aforementioned methods of alleviating AAAdisease in a subject the subject invention, the subject may be a mammal,for example, a human.

As used herein, the term “antigenic AAA protein” means any nativeprotein of human aorta which causes AAA disease.

Tolerance has been induced in humans to specific autoimmune antigenproteins by administering the antigen orally to the human subject. DavidTrentham, et al. report they have significantly reduced RheumatoidArthritis (RA) patients' symptoms by feeding them type II collagen, aprotein common in joint cartilage and a possible target of theautoimmune attack in RA (Trentham, D, et al., 1994). Their approach,called oral tolerization, takes advantage of a trick used by the bodythrough the digestive system suppress immune responses to those proteinsinstead of triggering them. Oral tolerization attempts to reduceautoimmune attacks by feeding the patients proteins—collagen, in thiscase—that are found at the site of autoimmune disease and that may havetriggered the autoimmunity in the first place (Barinaga, M., 1994).

In the aforementioned methods of the subject invention for alleviatingAAA disease in a subject, the subject may be a mammal, such as a human.

The subject invention also provides a recombinantly produced humanaortic protein which is immunoreactive with AAA-associatedimmunoglobulin. In one embodiment of the subject invention, therecombinantly produced protein is approximately 40 kDa.

This invention further provides a method of diagnosing AAA disease in asubject which comprises: (a) obtaining a suitable sample from thesubject; (b) contacting the sample with the recombinantly producedprotein of the subject invention under conditions permitting the proteinto bind to AAA-associated immunoglobulin if present in the sample; and(c) determining the presence of bound protein, the presence of boundprotein being indicative of AAA disease, thereby diagnosing AAA diseasein the subject. Methods of determining the presence of bound protein arewell known in the art as discussed above.

In the aforementioned method of the subject invention for diagnosing AAAdisease using the recombinantly produced proteins, the subject may be amammal, for example, a human.

This invention further provides a pharmaceutical composition foralleviating AAA disease in a subject which comprises the recombinantlyproduced protein of the invention and a pharmaceutically acceptablecarrier. The pharmaceutical composition may be constituted into any formsuitable for the mode of administration selected. Compositions suitablefor oral administration include solid forms, such as pills, capsules,granules, tablets, and powders, and liquid forms, such as solutions,syrups, elixirs, and suspensions. Forms useful for parenteraladministration include sterile solutions, emulsions, and suspensions.

This invention provides a method of alleviating AAA disease in a subjectwhich comprises administering to the subject an amount of theaforementioned composition comprising the invented recombinantlyproduced protein, said amount being effective to induce tolerance toantigenic AAA protein in the subject. Said method of alleviating AAAdisease in a subject comprises in one embodiment orally administering anoral composition according to the subject invention to the subject. Suchadministration imparts oral tolerization to the antigenic AAA protein asdiscussed above to the subject.

In the aforementioned method of the subject invention for alleviatingAAA disease in a subject, the subject may be a mammal, such as a human.

Finally, this invention provides a kit for detecting the presence ofAAA-associated immunoglobulin in a sample, which comprises: (a) a solidsupport having a plurality of covalently linked probes which may be thesame or different, each probe of which comprises a human aortic proteinwhich is capable of binding AAA-associated immunoglobulin; and (b) ameans for determining the presence of AAA-associated immunoglobulinbound to the human aortic protein. In an embodiment of the kit, thehuman aortic protein is selected from any of the following proteins: theisolated protein of approximately 40 kDa which is purified from humanaortic tissue and immunoreactive with AAA-associated immunoglobulin; theisolated protein of approximately 40 kDa which is purified from humanaortic tissue and immunoreactive with AAA-associated immunoglobulinhaving the amino acid sequence set forth in SEQ ID NO:1; a recombinantlyproduced human aortic protein which is immunoreactive withAAA-associated immunoglobulin; a recombinantly produced human aorticprotein which is immunoreactive with AAA-associated immunoglobulin andis of a molecular weight of approximately 40 kDa; a purified recombinanthuman aortic protein of approximately 24 kDa-28 kDa having the aminoacid sequence set forth in SEQ ID NO:2 which is immunoreactive with bothAAA-associated immunoglobulin and human kappa immunoglobulin; a purifiedrecombinant human aortic protein of approximately 28 kDa having theamino acid sequence set forth in SEQ ID NO:3 which is immunoreactivewith both AAA-associated immunoglobulin and human kappa immunoglobulin;a purified recombinant human aortic protein of approximately 24 kDa-28kDa having the amino acid sequence set forth in SEQ ID NO:4 which isimmunoreactive with both AAA-associated immunoglobulin and human kappaimmunoglobulin.

In an embodiment of the kit the means for determining the presence ofAAA-associated immunoglobulin bound to the human aortic protein is asuitable detectable label. In another embodiment of the kit, thesuitable detectable label is radioactive isotope, enzyme, dye, biotin, afluorescent label or a chemiluminescent label.

This invention provides a kit for detecting the presence of an elevatedor abnormal level of human aortic protein in a sample, whichcomprises:(a) a plurality of covalently linked probes which may be thesame or different, each probe of which comprises any one of aAAA-associated immunoglobulin and a human kappa immunoglobulin which iscapable of binding human aortic protein; and (b) a means for determiningthe elevated or abnormal level of human aortic protein bound toAAA-associated immunoglobulin or human kappa immunoglobulin bycomparison to a normal level of human aortic protein. Methods ofmeasuring the levels of proteins in samples from a subject are wellknown to one of ordinary skill in the art.

This invention will be better understood from the “Experimental Details”section which follows. However, one skilled in the art will readilyappreciate that the specific methods and results discussed therein arenot intended to limit, and rather merely illustrate, the invention asdescribed more fully in the claims which follow thereafter.

FIRST SERIES OF EXPERIMENTS Experimental Details Example 1

Human aortic tissue was extracted for microfibrillar proteins accordingto the method of Prosser, et al.¹¹ In brief, the tissue was firstextracted in a phosphate buffer containing potassium chloride 0.6M. Theinsoluble pellet was treated with bacterial collagenase in Tris buffer.The final tissue extraction utilized guanidinium chloride 6M in buffercontaining dithiothreitol 50 mM and EDTA 2 mM. Gel slices containing theprotein of interest were digested with trypsin or Lys-C and amino acidsequences were determined in the Protein Chemistry Core Facility, HowardHughes Medical Institute, Columbia University (New York, N.Y.).

RESULTS

A 59 residue sequence of AAAP-40 as experimentally determined is shownin Table I. Alignment on MFAP-4 and MAGP-36 (bovine) is shown, alongwith homologous sequences from the alpha and beta chains of humanfibrinogen. Five and 11 residue sequences of AAA-40 are shown in TableII, in alignment with sequences from vitronectin, MFAP-4, MAGP-36,fibrinogen-beta (from its calcium binding domain), and two other calciumbinding proteins (myeloid calcium binding protein and bovine aggrecan).

DISCUSSION

Fibrinogen-like domains are well-known in the MAGP's.⁸ The sequence ofAAA-40 shown in Table I has regions of substantial homology withsequences in the alpha and beta chains of fibrinogen. Another sequencethat we have determined (data not shown) matches residues 283-292 in thegamma chain. Since the three fibrinogen chains are believed to have asingle ancestral gene, it would appear likely that AAAP-40 is related tothe common ancestor since it has motifs that are used in all threefibrinogen subunits.

Kobayashi, et al. noted that MAGP-36 has the property ofcalcium-binding, although a candidate site for the calcium-bindingdomain has not been proposed. Kielty and Shuttleworth have observed thatincubation of intact microfibrils with EDTA rapidly results in grossdisruption of microfibrillar organization, which can be reversed byreplacing calcium.¹² Since fibrillin has 43-EGF-like motifs with calciumbinding consensus sequences, and calcium has been proposed toorchestrate the assembly of tropoelastin to the microfibril and hold itin register for crosslinking,¹³ it is hypothesized herein that thecalcium-binding domain of AAAP-40 may play a role in calcium-dependentmicrofibril assembly in the aorta. When GenBank was searched forhomologies of AAAP-40 and MAGP-36, sequences were found incalcium-binding myeloid-related protein (>pir/A44111:# 144-154), thecalcium-binding domain of human fibrinogen-beta, and bovine aggrecan(>pir/A39808:# 59-66) that have similarities to MFAP-4, MAGP-36, andAAAP-40. Bold type is used in Table II to highlight residues that appearto be conserved, with possible significance for the calcium-bindingfunction.

Another matrix protein detected in human embryonic tissue (sulfatedprotein 30 kDa=SP-30) has been reported to be immunoreactive withmonoclonal antibodies against human vitronectin,¹⁴ A sequence of AAAP-40that matches residues #230-240 in human vitronectin is also shown inTable II. Tomasini-Johansson, et al. proposed that SP30 is the humanhomolog of MAGP-31, but since MAGP-31 does not have a vitronectin-likedomain, SP-30 is more likely to be closely related to AAAP-40.

Finally, a brief comment on the nomenclature problem. Perhaps thesimplest approach for the present would be to assign the human MAGP's anumber in the order of their discovery. Thus, the first would be theprincipal component of the microfibril, fibrillin.

The second would be the protein of Smith Magenis syndrome, and the thirdwould be the protein described in the present application. MAGP-3 isprobably the human homolog of the bovine aortic protein of Kobayashi(MAGP-36), and the human homolog of Gibson's MAGP-31 has yet to beidentified. Notwithstanding the proposal of Zhao, et al, to call thisfamily of glycoproteins “MFAP”, 9 retention of the abbreviation MAGP isfavored, since the first bovine member of this family was so-named byGibson, et al. ten years ago and the term has been widely used eversince.

TABLE I This table shows sequence of AAAP-40, as determinedexperimentally. The sequence of AAAP-40 is aligned along a continuoussequence of MFAP-4, beginning at residue 140. Homologous regions ofMAGP-36 (bovine) and fibrinogen alpha (residues 120-132) and betaresidues 338-353 (human) are also shown. “( )” is used to designate anambiguous residue; “.” denotes a non- conserved residue; and “!” denotesa tryptic cleavage site.         ! (Y)F P(F)V D L M V M A N Q P MAAAP-40 122 T L K Q K  Y E L R V D L E D F E N N T A MFAP-4 T L   L K  YE L R V D L E D F E X N T A MAGP-36 ::      L R V E L E D . A . N . AHum Fib-a G E Y Y  D F F Q Y T X G M A K E Y D G F Q AAAP-40 142 Y A K YA  D F S I S P N A V S A E E D G MFAP-4 F A K Y A  D F S I S P N A V S AE E D G MAGP-36 Y T X G M  A K(I Y)A G N A L M D G A S G L M AAAP-40 162Y T L F V  A G F E D G G A G D S L S Y H MFAP-4 Y T L Y V  S G F E D G GA G D S L T Y H MAGP-36 Y . I . V  . K   T A G N A L Hum Fib-b

TABLE II Alignment of experimentally determined sequences of AAAP-40 onsequences from human vitronectin (VN, residues 230-240) and MFAP-4(beginning at residue #34). Alignments with other calcium-bindingproteins are shown, with the most highly conserved residues highlightedin bold type: Calcium- binding myeloid-related protein¹⁵ = CBP-M;Aggrecan (bovine)¹⁶ = Aggr; human fibrinogen beta (residues 144-157 fromcalcium- binding domain) = Fib-b. Q E L E K                        F E DG V L D P D Y P AAAP-40                                 R F E D G V L DP D Y P VN F C L Q Q P L D C D D I Y A Q G Y Q S D G VYL I Y P S MFAP-4S E L Q L P L D E D D I Y A Q G Y Q A D G VYL I   P S MAGP-36 T E L . .. L . E . D V Y . . . Y . . D CaBP-M           P . D E . D V Y Aggr S EL E K H Q L . . D . T Fib-b

Example 2 METHODS

Human subjects: Specimens of AAA tissue and peripheral blood were takenfrom patients at the time of infrarenal AAA repair. Normal abdominalaorta was harvested from organ donors. The protocols for humaninvestigation have been approved by the Institutional Review Board.Specimens were frozen at −110° C. immediately until use in biochemicalstudies. Serum was extracted from blood, and stored at −20° C. untilfurther studies.

Purification of IqG: Human IgG was purified from aortic tissue ard fromserum as described previously.¹¹ Briefly, the tissue was homogenized insalt buffer (2 M NaCl, 50 Mm TrisHCl, pH 7.5, 0.02% Na Azide). Thehomogenate was then centrifuged at 10,000 g for 1 hour at 4° C. IgG wasextracted from the supernatants using protein A-sepharose (Sigma, St.Louis, Mo.) column chromatography, and was eluted with 0.1M citric acidat the following pH; 3.0, 4.5 and 6.5. Each fraction was dialyzedagainst phosphate-buffered saline (PBS) at pH 7.5. Protein concentrationwas determined for each sample using dye-binding assays from Bio-RadLaboratories (Richmond, Calif.).

PBS tissue extraction: Each tissue sample was minced and washed severaltimes in PBS buffer containing 2 mM phenylmethylsulfonyl fluoride (PMSF)and 2 mM Na azide. Then the samples were homogenized in PBS buffer(described above) with 0.5 M ethylenediaminetetraacetic acid (EDTA). Thehomogenate was centrifuged at 650 g for minutes at 4° C., and thenultrafuged at 20,000 g for 1 hour at 4° C. The protein concentration foreach sample was determined by dye-binding methods. The samples werefrozen at −20° C. until further experiments.

Microfibril-associated glycoirotein (MAGP) tissue extraction: MAGP fromAAA tissue was extracted as described by Prosser et al.² Samples werehomogenized with buffer containing 0.1 M epilson-amino caproic acid, 2mM N-ethylmaleimide, 1 mM PMSF, and 0.1% Na azide. First, the sampleswere extracted in PBS with 0.6 M KCl. Next, the insoluble pellet wastreated with bacterial collagenase in Tris buffered saline (TBS). Thepreparation was then extracted with 6 M guanidine hydrochloride buffercontaining 50 mM dithiothretol and 2 mM EDTA.

Lymphocvte harvesting: Human peripheral blood lymphocytes were harvestedusing Ficoll-Hypaque gradient (Histopaqus>, Sigma, St. Louis, Mo.) asper standard protocol.

Immunohistochemistry: Tissue samples were fixed in formalin, embedded inparaffin, and sectioned at 6 um. The sections were deparaffinized inxylene and rehydrated using an ethanol step gradient.Iron-hematoxylin-von Gieson staining was performed using standardprotocols. The sections were blocked with 1% bovine serum albumin(Sigma, St. Louis, Mo.). Purified IgG(1:50), as described above, wasused as the first antibody. Alkaline phosphate-conjugated (APC) rabbitanti-human IgG (Sigma, St. Louis, Mo.) 1:50 was used as the secondantibody. The sections were washed, and then developed with VectastainBCIP/NBT kit (Vector laboratories, Burlingame, Calif.). After counterstaining with Fast Red (Sigma, St. Louis, Mo.), sections were dehydratedand mounted in Permont (Fisher, Pittsburgh, Pa.). Slides were examinedby light microscopy.

Modified Gomori Aldehyde Fuchsin-Peracetic Acid Reaction: The basicmethod for staining microfibrils was described by Fullmer et al.¹²Briefly, the 6 um paraffin-embedded tissue sections ere oxidized inperacetic acid for 15 minutes after deparaffinization. The sections werestained in Gomori's aldehyde fuchsin followed by hematoxylin (Sigma, St.Louis, Mo.). After counter stained with modified Halmis's solution, thesections were dehydrated and mounted.

Western Immunoblotting: Soluble protein in aliquots of PBS tissueextracts and MAGP tissue extracts, and harvested lymphocytes wereseparated by SDS/PAGE (12.5%), and then electroblotted ontonitrocellulose membranes. The membranes were blocked with 5% dry milk inPBS prior to incubating with the first antibody. Either purified AAAwall IgG (1:100), AAA serum IgG (1:100) or rabbit anti-human vitronectin(1:10,000) was used as the first antibody and allowed to incubateovernight at 4° C. After washing the membranes, either PC rabbitanti-human IgG (1:1000) or PC goat anti-rabbit IgG (1:5000) was used asthe second antibody. Immunoreactivity was detected by the VectastainBCIP/NBT color developing system.

Glycoprotein Detection: PBS tissue extracts and standard glycoproteinswere separated by SDS/PAGE (12.5%), followed by nitrocellulose membranetransfer as described above. Glycoprotein detection was carried outusing DIG™ glycan differentiation kit (Boehringer Mannheim Biochemica,Indianapolis, Ind.). Images of the membranes were digitized, enhancedand printed by computer.

Construction of cDNA Libraries: mRNA from aortic adventitia was reversetranscribed for insertion into the phagemid, Uni Zap XR™)lambda vectorsystem, in collaboration with Stratagene (La Jolla, Calif.). This systemaccommodates DNA inserts up to 10 kb in length.

Expression of cDNA: The phagemid from Uni-Zap XR vector was transfectedinto a strain of E. Coli (XL1-Blue MRF′, Stratagene™). The transfectedcells were plated on top agar, and then allow to grow at 45° C. untilsmall plaques were visible (approximately 4 hours). Nitrocellulosemembranes, impregnated with 10 mM isopropylthio-beta-D-galactopyranoside (IPTG), were placed onto the agar, andwere allowed to incubate for 4 hours at 37° C. The membranes wereremoved and blocked with 5% milk in TBS fro 45 minutes. Incubation wascontinued with rabbit anti-human vitronectin antibody (1:10,000) for 3hours at room temperature. The membranes were washed in TBS, andincubated with APC goat anti-rabbit IgG (1:5000) for 2 hours at roomtemperature. Following a series of washes in TBS, the membranes weredeveloped by Vectastain NBT/BICP color reagent system. The positiveplaques were rescreened-to obtain pure clones.

RNA was purified from a specimen of human aortic adventitia and reversetranscribed for insertion into the phagmid UniZap XR (Strategene™). Astrain of E. Coli, engineered for expression (XL1-Blue MRF—Stratagene™,was transfected. Human AAA wall and serum IgG's were used to localizethe autoantigen immuno-histochemically, and the Gomori aldehydefuchasin-peracetic acid reaction was used to evaluate co-distributionwith the elastin-associated microfibril. A glycan differentiation kitwas used to determine whether AAAP-40 is a glycoprotein.

RESULTS

Tissue sections incubated with AAA wall and serum IgGs showedimmunoreactive material co-localizing with elastin-associatedmicrofibrils, surrounding the birefrengent elastic fibers in the aorticadventitia (FIG. 1). Localization of elastin-associated microfibrils wasconfirmed by the modified Gomori reaction (Data not shown).

Western immunoblotting showed immunoreactivity of IgGs from AAA wall andserum with soluble aortic matrix proteins (FIG. 2). Uniqueimmunoreactive bands were detected in both AAA PBS and MAGP tissueextracts at MW 40 kDa. Rabbit anti-vetronectin antibody showed uniqueimmunoreactivity at MW 40 when incubated with AAA PBS tissue extracts(FIG. 3).

The glycan detection kit confirmed the presence of glycoprotein in AAAPBS tissue extracts at multiple molecular weights, including 40 kDa(FIG. 4). The 40 kDA band (AAA-40) had negative reactions to galanthusnivalis agglutinin (GNA) and sambucus nigra agglutinin (SNA), andpositive reactions to maackia amurensis agglutinin (MAA), peanutagglutinin (PNA), and datura stramonium agglutinin (DSA).

Positive expression of AAAP-40 was detected by antibody screening.Estimated frequency of the positive clones was 1:10,000 on primaryscreening and 1:1000 on secondary screening.

DISCUSSION

Chronic inflammatory cell invasion has been noted in AAAwall.^(13,14,15,16) Tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and IL-8 have been shown to be elevated in AAA tissueextracts when compared to control aortic tissue extracts.^(17,18,19)Elevated amounts of matrix-degrading enzymes have also been found in AAAwall associated with mononuclear cell infiltration.^(20,21,22) Theseobservations suggest that an autoimmune disease process plays a role inthe pathogenesis of AAA.^(13,14,17,23,24,25)

It has been reported that IgG from the aortic wall of patients withabdominal aortic aneurysms (AAA) is immunoreactive with a human aorticprotein (MW-80 kDa) that has features of the bovine aortic protein ofKobayashi et al. (MAGP-36).⁸ Since MAGP-36 occurs in nature as adisulfide dimer, the putative 80 kDa protein in aortic matrix may existas the dimeric form of AAAP-40.

The present studies demonstrate co-localization of AAAP-40. Glycandifferentiation analysis based on the findings shown in FIG. 4, suggestsAAAP-40 is a glycoprotein containing the following carbohydratesmoieties; sialic acid terminally linked alpha (2-3) to galactose,galactose beta (1-3)-N-acetylgalactosamine, and galactose beta(1-4)-N-acetyl galactosamine.

It is well known that human vitronectin is produced by the liver, andchat circulating vitronectin has MW 75 kDa.²⁶ The cleavage products areMW 65 and 10 kDa. AAA tissue extracts have unique immunoreactivity withrabbit anti-human vitronectin antibody at MW 40 kDa. Thus, clonespositive for reactivity with anti-human vitronectin antibody (in thecDNA expression library) are expressing AAAP-40 protein. The cDNAsequence of AAAP-40 will be further investigated using the positiverecombinants.

These present findings have diagnostic and therapeutic implications.Screening tests could be developed to identify circulating autobodies inthe peripheral blood from AAA patients. AAA disease progression could befollowed by peripheral blood antibody titers. Tolerance to theautoantigen may be induced in the AAA patient, and the furtherprogression of AAA disease may be prevented.

FIRST SERIES OF EXPERIMENTS References

1. Greenlee T K, Jr., Ross R., Hartman J L. The fine structure ofelastic fibers. J. Cell Biol 1966; 30; 59-71.

2. Prosser I W, Gibson M A, Cleary E G. Microfibrillar protein fromelastic tissue: a critical evaluation. Aust. J. Exp. Bio. Sci. 1984; 62:485-505.

3. Gibson M A, Huges J L, Fanning J C, Cleary E G (1986)o The majorantigen of elastin-associated microfibrils is a 31 kDa glycoprotein. JBiol Chem 261: 11429-11436.

4. Gibson M A, Sandberg L B, Grosso L E, Cleary E G (1991) ComplementaryDNA cloning establishes microfibril-associated glycoprotein (MAGP) to bediscrete component of the elastin-associated microfibrils. J Biol Chem266: 7596-601.

5. Bashir M M, Abrams W R, Rosenbloom J, Kucich U, Bacarra M, Han M-D,Brown-Augsberger P, Mecham R. Rosenbloom J (1994) Microfibril-associatedglycoprotein: characterization of the bovine gene and of therecombinantly expressed protein. Biochemistry 33: 593-600.

6. Kobayashi R, Mizutani A, Hidaka H (1994) Isolation andcharacterization of a 36-kDa microfibril-associated glycoprotein by thenewly synthesized isoquinolinesulfonamide affinity chromatography.Biochem Biophys Res Communic 198: 1262-6.

7. Zhao A, Lee C-C, Jiralerspong S, Juyal R C, Lu F, Baldini A,Greenberg F, Caskey C T, Patel P I (1995) The gene for a humanmicrofibril-associated glycoprotein is commonly deleted in Smith-Magenissyndrome patients. Human Mol Genetics 4: 589-597.

8. Tilson M D (1995). Similarities of an autoantigen in aneurysmaldisease of the human abdominal aorta to a 36-kDa microfibril-associatedbovine aortic glycoprotein. Biochem Biophys Res Communications 213:40-43.

9. Xia S., Ozsvath K., Hirose H. Tilson M D. Partial Amino Acid SequenceOf A Notice 40 kDa Human Aortic Protein, with Vitronectin-like,Fibrinogen-like, and Calcium binding Domains: Aortic AneurysmAssociated-Protein-40 (AAAP-40) Human MAGP-3), Proposed. Biochem BiophysRes Commun in press.

10. Tomasini-Johansson B R, Ruoslahti E, Pierschbacher M D 91993). A 30kDa sulfated extracellular matrix protein immunologically crossreactivewith vitronectin. Matrix 13: 203-214.

11. Gregory A K, Yin N X, Capella J., Xia S., Newman K M, tilson M D.Feature of autoimmunity in the abdominal aortic aneurysm. Arch Surg.1996: 23-25.

12. Fullmer H M, Lillie R D. The oxytalan fiber. A previouslyundescribed connective tissue fiber. J. Hisochem Cytochem 1958; 6: 425

13. Brophy C M, Reilly J M, Smith G J W, Tilson M D. The role ofinflammation in nonspecific abdominal aortic aneurysm disease. Ann VascSurg 1991; 5:229.

14. Koch A E, Haines G K, Rizzo R J, et al. Human abdominal aorticaneurysms: Immunophenotypic analysis suggesting an immune mediatedresponse. Am J Pathol 1990; 137: 1199-1219.

15. Beckman E N. Plasma cell infiltrates in athrosclerotic abdominalaortic aneurysm. Am J Clin Path 1986; 85:21-24

16. Rizzo R J, McCarthy W J, Dixit S N, et al. Typing of collagen andcontent of matrix protein in human abdominal aortic aneurysms. J VascSurg 1989; 10: 365-373

17. Newman K M, Jean-Claude J, Li H, Ramey W G, Tilson M D. Cytokinesthat activate proteolysis are increased in abdominal aortic aneurysms.Circulation 1994; 90: II-224-II-227.

18. Szekanecz Z, Shar M R, Pearce W H, Koch A E. Human atheroscleroticabdominal aortic aneurysms produce interleukin (IL)-6 andinterferon-gamma but not IL-2 and IL-4; the possible role IL-6interferon-gamma in vascular inflammation. Agents & Actions 1994;42:159-162

19. Koch AE, Kunkei SL, Pearch WH, et al. Enhanced production of thechemotactic cytokines interleukin-8 and monocyte chemoattractantprotein-1 in human abdominal aortic aneurysms. Am J Path 1993; 142:1423-1431.

20. Newman K M, Malon A M, Shin R D, et al. Matrix metalloproteinases inabdominal aortic aneurysm: Characterization, purification and theirpossible sources. Connective Tissue Res 1994; 30: 265-276

21. Newman K M, Jean-Clause J, Li H, Scholes J V, Ogata Y, Nagase H,Tilson, M D. Cellular localization of matrix metalloproteinases in theabdominal aortic aneurysm wall. J. Vasc. Surg 1994; 20: 814-820

22. Thompson R W, Holmes D R, Mertens R A, et al. Production andlocalization of 92 kilodalton gelatinase abdominal aortic aneurysms. Anelastolytic metalloproteinase expressed by aneurysm-infiltratingmacrophages. J Clin Invest 1995; 96: 318-326

23. Pearce E H, Sweis I, Yao JUST, McCarthy W J. Interleukin-lbeta andtumor necrosis factor-alpha release in normal and diseased humaninfrarenal aortas. J Vasc Surg 1992; 16: 784-789.

24. Jean Claude J, Newman K M, Li H, Gregory A K, Tilson, M D. Possiblekey role for plasmin in the pathogenesis of a abdominal aorticaneurysms. Surg 1994; 116: 472-475.

25. Reilly J M, Sicard G A, Lucore C L. Abnormal expression ofplasminogen activators in aortic aneurysmal and occlusive disease. J.Vasc Surg 1994; 19: 865-872

26. Preissner K T, Jenne D. Structure of vitronectin and its biologicalrole in haemostasis. Thrombosis and Haemostasis 1991; 66: 123-132

SECOND SERIES OF EXPERIMENTS Experimental Details

The partial amino acid sequence of a human microfibrillar glycoprotein˜40 kDa, which is called Aortic Aneurysm-associated Antigenic Protein-40kDa (AAAP-40) has been reported.(1) It has sequence homologies withvitronectin (VN) and fibrinogen (FB). A cDNA library was prepared frommRNA purified from human aortic adventitia. Since VN and FB are notsynthesized in aorta, the library was screened with polyclonalantibodies to VN and FB. The hypothetical proteins of clones 1 and 5share a novel domain structure and are the subject of thiscommunication.

METHODS

mRNA from aortic adventitia was reverse transcribed and inserted intothe phagemid, Uni Zap XR™ lambda, by arrangement with Stratagene (LaJolla, Calif.). The phagemid was transfected into E Coli (XL1-Blue MRF′,Stratagene™) The cells were plated on top agar and grown at 42° C. untilsmall plaques were visible. Nitrocellulose membranes impregnanted with10 mM isopropyl thio-beta-D-galactopyranoside were placed on the agarand incubated for 6 hours at 37° C. The membranes were removed andblocked with 5% milk in TBS for 45 minutes. Incubation was continuedwith either rabbit anti-human vitronectin (Sigma™ St. Louis, Mo.)(1:10,000) or fibrinogen antibody (Sigma™ 1:2,500) for 2 hours at roomtemperature. The membranes were washed in TBS and incubated withalkaline-phosphatase-conjugated goat anti-rabbit IgG (1:5000) (Sigma™)for 2 hours at room temperature. Following a series of washes in TBS,the membranes were developed by the Vectastain NBT/BICP color reagentsystem. The positive plaques were rescreened to obtain pure clones.

Excision from positive clones was carried out using the Ex Assist/SOLRSystem (Strategene™) The phages were extracted and re-transfected toSOLR cells with amplified Ex Assist Helper Phage (Strategene™). Thesecells were grown overnight at 37° C., on ampicillin-supplemented medium,which inhibits the growth of non-transfected cells. The cells were thenharvested and lysed by alkali buffer, and the DNA was purified byphenol-chloroform extraction. DNA sequencing was carried out by the corelaboratories at Columbia College of Physicians and Surgeons, ColumbiaUniversity, New York, N.Y.

Immunohistochemistry was performed with rabbit anti-human Ig kappa,1:50, (Sigma™) and APC goat anti-rabbit IgG, 1:50, whole molecule,(Sigma™). APC goat anti-human Ig heavy chain (FC'specific, Sigma™),1:150, was used for control experiments. The slides were developed withBCIP-NBT kit (Biomeda, Foster City, Calif.) and counterstained with FastRed (Sigma™).

RESULTS

The proteins encoded by clones 1 and 5 have similar features and domainstructures, which can be aligned in eight regions beginning with theN-terminus (Tables III and IV). Region 1 is a lengthy sequence highlyhomologous to Ig kappa V (88-120 residues). Region 2 is a 44-63 residuesequence, which in the case of clone 5 has a six residue sequence thatalso occurs in cytomegalovirus. Region 3 is a 9-12 residue sequence thatis conserved from Ig-kappa. Region 4 is a possible calcium-binding motifin clone 1, while clone 5 has a gly/pro rich sequence. Region 5 is anaromatic-rich sequence. Region 6 is a gly/pro rich sequence in bothclones. Region 7 returns to a conserved sequence from Ig kappa, which inclone 1 contains an RGE motif. Region 8 is a C-terminal sequence of68-70 residues containing a second aromatic motif.

Immunohistochemistry revealed binding of rabbit anti-human Ig kappaantibody to the aortic adventital microfibril, in specimens of bothnormal and aneurysmal human abdominal aorta.

DISCUSSION

Two of five clones, selected from an expression library of aorticadventitia, encode unique proteins sharing sequences of Ig kappa,gly/pro rich (collagenous) motifs, and aromatic motifs that occur inseveral proteins of the extracellular matrix. Both proteins have asimilar domain structure with at least 8 regions: 1) Ig kappa (847120residues in length); 2) ser/thr-rich motif (44-63); 3) a second Ig kappamotif (9-12); 4) either a possible calcium-binding motif or a gly/prorich sequence (35-43); 5) an aromatic rich sequence (6-7); 6) anothergly/pro rich sequence (62-72); 7) a third Ig kappa sequence (26-28); and8) a C-terminal 68-70 residue sequence with another aromatic motif.

These are novel proteins. The use of an immunoglobulin domain as aspecificity determinant has been described in the fibroblast growthfactor receptor,(2) but the use of Ig kappa sequences in matrix proteinshas not been described. The gly/pro rich sequences are typical of thecollagens. The aromatic motifs resemble similar motifs in vitronectin(VN: FFFS), microfibril-associated protein 4 (3) (MFAP-4: FYYS), andAAAP-40 (FFYS and YY.FFQYT). (1)

GenBank searches have led to the conclusion that aromatic motifs,followed by SP, TS, or L, are rare in structural mammalian proteins,except for the above-mentioned. FFFSP occurs in cytomegalovirus (CMV)(4), as does the sequence CRIKN.AV in clone 5, consistent with thehypothesis that the clinical association of aneurysm disease andinfection with CMV (5) may be on the basis of molecular mimicry.(6) FFFLis limited to a human T-cell receptor, a secretory product in mouse, andherpes virus 2 @ residue 62. The herpes virus is interesting, because itis another potential molecular mimic.(6, 7) FFTS is reported only in oneother protein, lens fiber major protein.(8) Another interesting featureof clone 1 is the possible calcium-binding sequence. The importance ofcalcium in the self-assembly of the microfibril has been documented,(9)and we have previously reported a possible calcium-binding motif inAAAP-40.(1) MAGP-36 is also a calcium-binding microfibrillar protein,with tissue distribution limited to the aorta in pig.(10)

Because of these unique features, we suggest the name “kappafibs” forthese hypothetical proteins, to reflect their use of Ig kappa motifs andtheir possible role as structural elements of the microfibril.

TABLE III Clone 1. Amino acid sequence of the hypothetical proteinencoded by clone 1. “*” = cystein residue used in disulfide bond of Igkappa, present (along with the following R.SQ) in both clones. “X” =indeterminant residue.                                             *MDMRVPAQLLGLLLLWLPGARCDIQLTQSPSLLSASVGDRVMITCRASQA 50-clone 1MDMRVPAQLLGLLLLWLPGARCAIRIAQSPSSLSASTGDRVTITCRASQG Ig-k (11) <<--FirstIgk homology region begins at #1---------  ..........LLLWI.GA..DI..TQSP.L..VS.GERATINCRSSQ clone 5        LLAL.LL.L MFAP - 4ISSFLAWYQQKPGKAPKLLIHAASSLQTGVPSRFSGSGSGTXFTLTISXL 100-clone 1ISNYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRL Ig-k (12) ------FirstIgk homology region continues---------QSEXLQLYYCQHLKGYPITFRPRDTXGXXXNCXCTIXSSSSRHLXNIEIW 150-clone 1EPEDFAVYYGQQYGSSPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK-- Ig kappa -First Igkends---->   (asparagine/ser/thr/rich)                                   --->13<---XCLCCXACYXITSXPKKAKFHWKVDNPSNRVTPQKNFPXQKVFENFGOGKXG 202-clone1SGTASVVGLLNNFYPREAKVQWKVDNALQSGNSQESVT------------- Ig kappa            <2nd conserved Igk          RIFENY.KGRKG CaBnd(14)             motif in clone 1>          K.YD..G.GQ Cabnd(15)                                        KV.E.F..G Cabnd(16)                                        KLYE.FED Cabnd(17)  XKGXGXXXXFFFXPFGXXXXFGXXCXCWXPGXXKIFXXPGGAKVqGEG 250-clone 1 --Ig kcontinues directly from VT above to EQ below--        FirstAromatic               Gly/Pro rich            Motif                       MotifGKXLPIGXFPXECXQSXTARTALTASAAPTRKHKVYAKEVTHQGL--PVTKSXNRGE 307-cl--------EQDSKDSTYSLSSTLTLSKADYEKHKVYAGEVTHQGLSSPVTKSFNRGE IgKappa                             <3rd conserved Igk motif>                          S.ADY...K AAAP80 RGD VNCXXREKCPHXXXSSSLTPSHPLAXXDPFSTGDLPLLRSSSXFFTSPPSSS 357-clone 1                                   <2nd Aromatic motif                                     also ser/thr rich>            SSSL           FST.D MFAP-4                            TST.DAAAP-80                             STGDIPML...S FGF-R(18)                             FST  VPL.RT HeparinB(19)                           TSTAD Fib-B (20)                                        FFFSP Cytomeg                                       FFFS VN                                       FFYS AAAP-40LAXIFALIMLMLEENEXIK     372# <end of clone 1>

TABLE IV Clone 5. Amino acid sequence of hypothetical protein of clone5.                                           *MVLQTQVFISLLLWISGANGDIVMTQSPDSLGVSLGERATINCRSSQRL 49 Clone 5MVLQTQVFISLLLWISGA+GDIVMTQSPDSLAVSLGERATINCKSSQSV IgK V-IV (21)<<--First Igk homology region begins at #1-------           LL.L.V           DSL V.L MFAP-4FFGSNSKNYLAWYQQKPGQSPKLLIYWASTRDSGVLTDSLAAGLGXI 96 Clone 5LYSSNNKNYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTD Ig kappa ----------FirstIgk motif ends----> SLSPSXXCRLKNLAILXLSAIIIISXXTFRPWGTXLXIQXKCWXAXIFXSF147 - Clone 5 FTLTISRLEPEDFAVYYGQQYGSSPLTFGGGTKVEIKRTVAA----PSVFI Igkappa SISP MAGP-36 SISP                LLLLS MFAP-4        CRIKN.AVCytomeg FPPXEKQFK-<----First gly/pro rich motif(22)------> 218 Clone 5FPPSDEQLK---------(joins EI below)--------------- Ig kappa <2nd Igk Motif> FFFFSPFLXGWXLGXLFXGPXEKIFFPXGPKKRGRGXKXPPNWGKSPSG 268 Clone5------------------EITASVVGLLNNFYPREAKVQWKVDNALQSG Ig kappa  <1staromatic <-----Second gly pro rich region begins-> motif>  FFFSPCytomegaly V  FYYS AAAP-40  FYYS MFAP-4  FFFS Vitronectin  FFF.PF Clone1 XXXGRGXQGKGNLKALWXEPXRLGKGGIRGXNKXXAXEVTHSGLSFAXSKKXXQGRX Clone 5NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAGEVTHQGLSSPVTKSFNRGEG Ig k   -G/Pmotif                   <3rd Igk Homology region> ends ------>          A.AA...K.KVYA.EVTH.GL....S....E Clone 1LEGEVPPPVXXXQPDPLPSFGLXPFFHRGXTPIXVXQXIFYXTPLXXLGFNYXNVXXXXINKVXFL                  <? 2nd aromatic motif>         <end of clone 5>                                                            #393

SECOND SERIES OF EXPERIMENTS References

1. Xia S, Ozvath K, Hirose H, Tilson M D. Partial amino acid sequence ofa novel 40 kDa human aortic protein, with vitronectin-like,fibrinogen-like, and calcium binding domains: aortic aneurysm-associatedprotein-40 (AAAP-40) [Human MAGP-3, proposed].

Biochem Biophys Research Communications, 1996; 219: 26-39.

2. Zimmer Y, Givol D, Yayuon A. Multiple structural elements determineligand binding of fibroblast growth factgor receptors: evidence thatboth Ig domain 2 and 3 define receptor specificity. J Biol Chem 1993;268: 7899-7903.

3. Zhao Z, Lee C-C, Jiralerspong S, Juyal R C, Lu F. Baldini A,Greenberg F, Caskey C T, Patel P I. The gene for a humanmicrofibril-associated glycoprotein is commonly deleted in Smith-Mayenissyndrome patients. Human Mol Genetics 1995; 4: 589-597.

4. Cytomegalovirus >sp¦P09727¦US11_HCMVA.

5. Tanaka S, Komori K, Okadome K, Sugimachi K, Mori R. Detection ofactive cytomegalovirus infection in inflammatory aortic aneurysms withRNA polymerase chain reaction. J Vasc Surg 1994; 20: 235-43.

6. Ozsvath KJ, Hirose H, Xia S, Tilson M D. Molecular mimicry in humanaortic aneurysm diseases. New York Academy of Science ConferenceAbstract Book: The Abdominal Aortic Aneurysm—Genetics, Pathophysiology,and Molecular Biology 1996 (Mar 7-9): p28.

7. DePalma R G, Sidaway A N, Giordana J M. Associated aetiological andatherosclerotic risk factors in abdominal aneurysms. in: The Cause andManagement of Aneurysms, ed. R M Greenhalgh, J A Mannick, J T Powell. WB Saunders Company, London 1990; 97-104.

8. Human lens major protein, >pir¦A22444

9. Kielty C M, Shuttleworth C A (1993). The role of calcium in theorganization of the fibrillin microfibrils. FEBS 336: 323-326.

10. Kobayashi R, Tashima Y, Masuda H, Shozawa T, Numata Y, Miyauchi K,Hayakawa T. Isolation and characterization of a new 36-kDamicrofibril-associated glycoprotein from porcine aorta. J Biol Chem1989; 264: 17437-44.

11. Ig kappa, X72444, is the best match with the hypothetical proteinencoded by the first 50 exons.

12. Ig kappa, pir>A23746=Ig kappa chain V-III.

13. Intervening sequence uncertain, since forward and reverse sequencesof the clone do not overlap:

14. Calcium-binding motif from 25 K calcium-binding protein—Tetrahymenathermophila >gi|161744.

15. Calcium binding protein—10 kDa—CB25_TETTH >sp|P09226

16. 55 kDa Calcium binding protein (spinach), residues 1-3 and 6-11,sp|P31806

17. Calmodulin domain, >gnl|PID|e237260.

18. Fibroblast growth factor receptor 2b, rat, >gi|1551272

19. Heparin-binding protein, 46 K—bovine: >pir||S09032.

20. Fibrinopeptide B FIBB_VULVU >sp¦P14482

21. A01902 <pir>=Ig kappa chain V-IV region, best match with first 50residues of clone 5.

22. The following 62 residues in clone 5 had many ambiguities, but ofthe 41 that were determined, 15 (36%) were either pro or gly. Thus, thisregion is similar to the gly/pro rich motif in clone 1.

THIRD SERIES OF EXPERIMENTS

Immunoglobulins (IgGs) isolated from serum and aortic wall of patientswith abdominal aortic aneurysms (AAAs) are immunoreactive with a 40 kDAaortic protein, which have been purified and partially sequenced: AorticAneurysm-associated Antigenic Protein-40 (AAAP-40) [^(1, 2, 3, 4)] Ithas sequence similarities to a protein with tissue distribution limitedto the aorta in pig: Microfibril-Associated Glycoprotein 36 kDa(MAGP-36). [^(2, 5, 6, 7)] Fibrinogen, vitronectin, and Ig kappa sharesequence similarities with AAAP-40 [^(8, 9)]. Pathogens associated withaneurysmal disease, including herpes simplex virus and cytomegalovirus[¹⁰], also have sequence similarities with AAAP-40. These viruses havebeen implicated in the pathogenesis of AAA disease by molecular mimicry[^(11, 12, 13)].

A cDNA library of mRNA from the aortic wall of a AAA patient wasscreened with rabbit anti-human vitronectin, yielding five positiveclones. Two clones (Clone 1 and Clone 5) have been found to encodeunique by hypothetical microfibrillar proteins (rAAAP-CL1 andrAAAP-CL5), with similar domain structure, including three motifs fromIg kappa (hence, “Kappafibs”). [^(6, 14)]. It has been shown inimmunohistochemical studies that both rabbit anti-human IgG kappa andAAA IgG are immunoreactive with proteins that co-distribute with aorticadventitial fibrils [^(1, 6)]. It is here reported that an expressionvector transfected with Clone 1 and Clone 5 synthesizes the recombinantproteins, rAAAP-CL1 and rAAAP-CLS, (detectable with AAA IgGs and rabbitanti-human Ig kappa antibody), and these gene products are furthercharacterized (including their tertiary structure and evolutionaryorigins), and it is proposed that the kappafibs may represent a novelfamily of matrix cell adhesion proteins (MAT-CAMS).

METHODS

Human subjects: At the time infrarenal AAA repair, tissue samples andblood samples were obtained from patients. Normal abdominal aortictissue was harvested from organ donors. The protocols for humaninvestigation have been approved by the Institutional Review Board.

Purification of IgG: Human IgG was purified from aortic tissue andperipheral blood samples as previously described.

Construction of cDNA libraries: mRNA from AAA adventitia was, reversetranscribed for insertion into the phagemid, Uni Zap XR™ lambda vectorsystem, by Stratagene™ (La Jolla, Calif.). DNA inserts up to 10 kb inlength are accommodated by this system.

Screening of cDNA: The phagemid from Uni Zap XR™ was transfected into anE. coli strain (XL1-Blue MRF′, Stratagene™. After plating thetransfected cells on top agar, the cells were incubated forapproximately four hours at 45° C., until small plaques were visible.Isopropyl thio-beta-D-galactopyranoside (IPTG, 10 mM) impregnatednitrocellulose membranes were placed onto the agar and allowed toincubate for four hours at 37° C. The membranes were removed and blockedfor 45 minutes with 5% milk in Tris buffered saline (TBS). Incubationwas continued for two hours with either rabbit anti-human vitronectin(Becton Dickerson Labware, Bedford, Mass.) 1:10,000, or fibrogenantibody (Dako, Denmark) 1:2,500 at room temperature. After a series ofwashes in TBS, the membranes were incubated with alkaline phosphataseconjugated (APC) goat anti-rabbit IgG (1:5,000) (Sigma) for two hours atroom temperature. Following a series of washes in TBS, the membraneswere developed with Vectastain NBT/BICP color reagent system (Vector,Burlingame, Calif.). The positive plaques were rescreened to obtain pureclones.

Purification and sequence of DNA: Excision from positive clones wascarried out using the Ex-Assis/SOLR System (Stratagene). The phages wereextracted and tranfected to the SOLR with Ex-Assist Helper Phage(Stratagene). These cells were grown overnight at 37° C., onampicillin-supplemented medium. The cells were harvested and lysed byalkali buffer, and the DNA was extracted by phenol-chloroform extractionmethods. DNA sequencing was performed by the Columbia University DNAsequencing laboratory, Columbia College of Physicians and Surgeons, NewYork, N.Y.

Expression of the recombinant DNA: The screened and purified plasmid wasmixed with XL1-Blue MRF′ cells (Stratagene) pretreated with CaCl₂. Themixture was heat shocked to allow transformation. The transformed cellswere allowed to grow in non-selective media, and were then plated ontoampicillin enriched agar. A single colony was harvested and grown inliquid media supplemented with 10 mM IPTG. The cells were then lysedwith lysozyme (Sigma, St Louis, Mo.), the debris was removed bycentrifugation. Dye binding assays were undertaken to determine theprotein concentration of the supernatant. A non-transfected colony wasprocessed to identify cellular proteins.

Western Immunoblots: Western immunoblots were probed with either rabbitanti-human Ig kappa antibody, APC goat anti-human heavy chain antibody,IgGs purified from the sera of AAA patients and control patients.

Computational biology and graphical displays: Protein sequences weredownloaded from Retrieve ¹⁵ at the National Institutes of Health.Multiple alignments were performed with SIM™ at the Biologist's ControlPanel of the Human Genome Project of the Baylor College of Medicine[¹⁶]. The probabilities of similarities between proteins was calculatedby Blast-PT™ and Motif™ [^(17, 18)]. Pairwise alignments were calculatedat the Biologists Control Panel and similarity plots were displayed byLalnView [¹⁹]. Protein modeling were performed by SwissModel, [²⁰] andthe output files were displayed by Rasmol [²¹]. The evolutionary treewas calculated based on PAM™ distances calculated by AllAll™ [²²]. FIG.11 is not precisely drawn to scale. In the cartoon mode of Rasmol thebeta strands are shown in yellow and the alpha coils are shown in red[¹⁹]. In “group” mode of Rasmol, color progresses from blue to darkgreen to light green to yellow to red as it reads from the N-terminus tothe C terminus of the protein.

RESULTS

Recombinant proteins were expressed by the XL1-Blue MRF′ cellstransfected with the cDNA of Clone 1 and Clone 5. Western immunoblotsshowed that rAAAP-CL1 and rAAAP-CL5 were immunoreactive with serum IgGsfrom AAA patients at a MW of 28 kDa (FIG. 6). Western immunoblots probedwith serum IgGs from normal volunteers showed no unique immunoreactivity(FIG. 7). rAAAP-CL1 and rAAAP-CL5 were immunoreactive on Westernimmunoblots probed with rabbit anti-human Ig kappa antibody (FIG. 8),not seen with APC goat anti-human heavy chain antibody (FIG. 9).

A search of the N-terminal 125 residues of rAAAP-CL1 with BlastPidentified IgK precursor KVlJ [²³] as the closest match (p=1.4×10⁻⁵⁰).FIG. 10A is a model of rAAAP-CL1 as calculated and displayed by RasMol,with beta-strands labeled according to the standard convention. [²⁴].FIG. 10B is the same molecule, rAAAP-CL1 with color coding blue to red,illustrating the progression from N-terminus to C-terminus. FIG. 10C isthe backside of rAAAP-CL1 showing the bottom sheet of the beta-sandwichwith strands c, c′, f and g.

Among the first 100 matches by BlastP for the Ig-like sequence ofrAAAP-CL1 (residues 20-124) was 1VCA [²¹] (immunoglobulin superfamily,integrin binding molecule of mouse, also known as VCAM1-mouse), withp=0.02. The homology occurred between rAAAP-CL1 and the first Ig-likedomain of VCAM-1 mouse. Ninety-two of the other 100 matches wereimmunoglobulins. FIG. 10D is a cartoon of the tertiary structure of thefirst Ig-like domain of VCAM-1 mouse.

FIG. 10E is a pair-wise comparison displayed by LalnView of the aminoacid sequence similarities of rAAAP-CL1 and VCAM-mouse (domains 1-7).Since cytomegalovirus has been recently implicated as a potentialmolecular mimic in AAA disease, an additional alignment of rAAAP-CL1with a cytomegalovirus protein, shows numerous similarities (FIG. 10F).

DISCUSSION

AAA wall has been found to have an infiltrate of chronic inflammatorycells [^(1, 25)]. Immunohistochemical studies using AAA IgG have shownthat binding co-localizes with adventitial elastin-associated fibrils.[¹] Purification and sequencing studies have lead to thecharacterization of a putative autoantigen (AAAP-40) with sequencehomologies to MAGP-36, vitronectin, fibrinogen and with severalpathogens associated with aneurysmal disease (Treponema pallidum, Herpessimplex virus and cytomegalovirus).(2)

An effort to clone the cDNA for AAAP-40 from an expression library madewith human aortic mRNA has resulted in the cloning of three relatedgenes, which encode recombinant proteins that are also immunoreactivewith AAA serum and tissue IgG. Two of the clones (Clone 1 and Clone 5)are similar to each other, and they also have even stronger similaritiesto the variable region of the immunoglobulin kappa (IgK's). [⁶]Accordingly, they have been called “kappafibs.”

The similarities of rAAAP-CLl and VCAM-1 mouse are evident on comparingFIGS. 10A and 10D. There is virtual identity in the predicted tertiarystructure of the front beta-sheet of rAAAP-CL1 and the first Ig-likedomain of VCAM-1 mouse (although the first Ig-like domain of VCAM-1lacks a backside beta-sheet). These similarities of spatial structureoccur despite a low level of conservation of amino acid residues asshown in FIG. 10E, where the similarities of amino acid sequence betweenthe first 124 residues of rAAAP-CL1 and VCAM-1-mouse are less than 30%,confirming the observation of Bork, Holm, and Sander that“three-dimensional structures are much more conserved in evolution thanare sequences”. [¹⁹].

Because the N-terminal portion of the CD loop (beginning with aglutamine residue) “appears to be vital in directly mediating integrinbinding”, [²⁶] we were interested to note that a glutamine residueoccupies a similar site in rAAAP-CL1, as well as in the BC C-C C′-C″ andFG loops.

It has been reported that kappafibs have similarities to the fibrinogensand to the immunoglobulins kappa [⁶]. Similarity to the fibrinogens wasnot unexpected, since the previously described microfibrillar proteins(AAAP-40 [²], MAGP-36 [ ]⁴ and MFAP-4 [ ]²)⁷ have fibrinogen-likemotifs. The strategy for seeking a candidate common ancestor was toperform a multiple alignment of four members of the overall family. Theprogram Motif provided a promising 40 residue sequence of similarity(Table V). A Blast-P search of the 40 residue sequence from MFAP-4(beginning FKR . . . ) revealed a highly significant alignment with anancient protein from the sea cucumber (p=2×10⁻¹¹], which is calledfibrinogen-related protein-A precursor [²⁸] (Table VI). A multiplealignment for all the proteins studied is shown in Table VII.

The similarities of the clones to the immunoglobulins occur in thevariable domains of the immunoglobulins sheet, suggesting that these newproteins may be tissue-specific signals that inform cells as to theirwhereabouts, e.g. Matrix Cell Adhesion Molecules, or “MAT-Cams”. Asmooth muscle cell, for example, residing in the aorta instead of theureter, needs some clues as to the appropriate ratio of Type III or TypeI collagen to manufacture to suit the requirements of its specificlocation. This function is perhaps more primitive than soluble immunity,so it is conceivable that the immunoglobulins arose from the MAT-Cams inmolecular evolution. Non-specific immunity first appeared in primitivemetazoans like sponges; but soluble immunity did not appear untilrelatively recently in evolutionary history (cartilagenous sharks).²⁹Considering that matrix proteins probably appeared with the firstmulticellular organisms, the notion that matrix adhesion molecules mayhave appeared before immunoglobdlins is not implausible.

Xu and Doolittle suggested that the mammalian fibrinogens share a commonancestor with the fibrinogen-like peptide of the invertebrateEchinoderm, which appeared in similarity searches.³⁰ The homologies ofthe fibrinogen-related protein-A precursor of sea cucumber span almost80% of the length of MFAP-4. To examine the hypothesis that MAT-Cams mayhave preceded soluble immunity, AllAll³¹ were used to calculate thepoint-accepted mutation rates (PAM units) for several proteins. Forfibrinopeptide related proteins, a PAM unit has been estimated to beabout 1 million years.³² The phylogenetic tree in FIG. 11 indicates thatclone 1 is significantly closer to an immunoglobulin kappa than anyother protein under consideration. FIG. 11 also suggests that there weretwo clusters of evolutionary activity. The first was around the time ofthe Cambrian radiation (producing the fibrinogens and themicrofibril-associated glyco-proteins); and the second much morerecently (spawning the CAM's first and then the immunoglobulins).

In conclusion, there appear to be several candidate autoantigens in AAAdisease. The two described here have lengthy immunoglobulin domains andmay serve as cell adhesion molecules. Finally, the novel hypothesis israised that the MAT-Cams may be closer to their common ancestor than thesoluble immunoglobulins.

TABLE V Alignment of a similarity block from Clone 5g, Ig kappa,MAGP-36, and MFAP-4 by the MOTIF program. Clone 5 57WYQQKPGKAPKLLIHAASSLQTGVPSRFSGSGSGTXFTLT Ig-kappa 57WYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLT MAGP-36 27SGPRFCGSVSFFRGWNDYKLGFGRADGEYWLGLQNMHLLT MFAP-4 82FQKRFNGSVSFFRGWNDYKLGFGRADGEYWLGLQNMHLLT

TABLE VI >1790817 (L38486) microfibril-associated glycoprotein 4[Homosapiens] Length = 256 Score = 125 (58.7 bits), Expect = 2.7a-11, P= 2.7a-11 Identities = 19/40 (47%), Positives = 30/40 (75%) SEA CUCUMBER 1 FQRRIDGTINFYRSWSYYQTGFGNLNTEFWLGNDNIHYLT  40    FQ+R +G+++F+R W+  Y+GFG  + E+WLG  N+H LT MPAP-4 83 FQKRTNGSVSFFRGWNDYKLGFGRADGEYWLGLQNMHLLT122

TABLE VII istalW Multiple Sequence Alignment Results 1            1516           30 31           45 1 IgK1 ---MKKTAIAIAVAL AGFATVAQAAELTQSPSSVS---ASVGDRV 2 clone_(—1) -MDMRVPAQLLGLLL LWLPGARCDIQLTQSPSLLS---ASVGDRV 3 Igk2 ---MVLQTQVFISLL LWISGAYGDIVMTQS PDSLA---VSLGERA 4clone_(—5) ---MVLQTQVFISLL LWISGANGDIVMTQS PDSLG---VSLGERA 5 magp_(—36)--------------- --------------- --------SELQLPL 6 mfap_(—4)---MKALLALPLLLL LSTPPCAPQVSGIRG DALER---FCLQQPL 7 sea_(—cuc)MFSFIMKAAILLILV GCISFCISSEPLNES EITFEREERSLADPA 8 aaap_(—40)--------------- --------------- --------------- 46           6061           75 76           90 1 IgK1 T-------ITC--RA SQGISS-----------------WLAWYQQ 2 clone_(—1) M-------ITC--RA SQAISS-----------------FLAWYQQ 3 IgK2 T-------INC--KS SQSLLYS-------- ---SNNKNYLAWYQQ 4clone_(—5) T-------INC--RS SQRLFFG-------- ---SNSKNYLAWYQQ 5 magp_(—36)D-------EDD--IY AQGYQA--------- --------DGVYLIP 6 mfap_(—4)D-------CDD--IY AQGYQS--------- --------DGVYLIY 7 sea_(—cuc)GRQKRQSGLSCPKRI SHSPEYPRDCYDILQ SCSGQSPPSGQYYIQ 8 aaap_(—40)--------------- --NENN--------- --------VVNEYSQ 91          105106         120 121         135 1 IgK1 KPGKAPKLLIYSASS LQSG----VPSRFSGSGSG-TDFSLTISSL 2 clone_(—1) KPGKAPKLLIHAASS LQTG----VPSRFSGSGSG-TXFTLTISXL 3 Igk2 KPGQPPKLLIYWAST RESG----VPDRFSG SGSG-TDFTLTISSL 4clone_(—5) KPGQSPKLLIYWAST RDSG----VLTDSLA AGLGXISLSPSXXCR 5 magp_(—36)-SGP----------- -----------RFCG SVSFFRGWNDYKLGF 6 mfap_(—4)PSGPSVPVPVFCDMT TEGGKWTVFQKRFNG SVSFFRGWNDYKLGF 7 sea_(—cuc)PDGGN-LIKVYCDME TDEGGWTVFQRRIDG TINFYRSWSYYQTGF 8 aaap_(—4O)ELEKX---------- FEDG-------VLDP DYPX---WTVFQXYF 136         150151         165 166         180 1 IgK1 QPEDSATYYCQQANS FPYTFG----QGTKVEIKRTVAAPSVFIFP 2 clone_(—1) QSEXLQLYYCQHLKG YPITFRPRDTXGXXXNCXCTIXSSSSRHLX 3 Igk2 QAEDVAVYYCQQYYS TPPMFG----QGTKV EIKRT---------- 4clone_(—5) LKNLILXLSAIIIIS XXTFRP----WGTXL XIQXKCWXAXIFXSF 5 magp_(—36)GRADGEYWLGLQNMH LLTLK--YELRVDLE DFEXNTAFAKYADPS 6 mfap_(—4)GRADGKYWLGLQNMH LLTLKQKYELRVDLE DFENNTAYAKYADFS 7 sea_(—cuc)GNLNTEFWLGNDNIH YLTSQG---DYELRV ELNNTLGNHYYAKYN 8 aaap_(—40)PFVDLMVMANQPMGE KYYDF-------FQY TXGMAKEYDGFQYTX

THIRD SERIES OF EXPERIMENTS References

1. Gregory, A. K., Yin N. X., Capella, J., Xia, S., Newman, K. M., andTilson, M. D. Features of AutoImmunity in the Abdominal Aortic Aneurysm.Arch of Surg. 131:23-25, 1996.

2. Xia, S., Osvrath, K. J., Hirose, H., and Tilson, M. D. Partial AminoAcid Sequence of a Novel 40 kDA Human Aortic Protein withVitronectin-like, Fibrinogen-like, and Calcium Binding Domains: AorticAneurysm-Associated Protein-40 [Human MAGP-3, Proposed]. Biochem.Biophys. Res. Commun. 219, 36-39, 1996.

3. Tilson, M. D. Similarities of an Autoantigen in Aneurysmal Disease ofthe Human Abdominal Aorta to a 36-kDa Microfibril-Associated BovineAortic Glycoprotein. Biochem. Biophys. Res. Commun. 213:40-43, 1995

4. GeneBank Accession Number SP¦P80520

5. Kobayashi, R., Mizutani, A., Hidaka H. Isolation and Characterizationof a 36 kDA Microfibril Associated Glycoprotein by the Newly SynthesizedIsoquinolinesulfonamide Affinity Chromatography. Biochem. Biophys. Res.Commun. 198:1262-1266, 1994.

6. Kobayashi, R., Tashima, Y.; Masuda, H., Shozawa, T., Numata Y.,Miyauchi, K., and Hayakawa, T. Isolation and Characterization of a New36-kDa Microfibril-Associated Glycoprotein from Procine Aorta J. Biol.Chem. 264:1737-44, 1989.

7. GeneBank Accession Number GI¦543129

8. GeneBank Accession Number SP¦P19477

9. Ozsvath, K. J., Xia, S., Hirose, H., Tilson, M. D. Two HypotheticalProteins of Human Aortic Adventitia, with Ig Kappa, Collagenous, andAromatic-Rich Motifs. Biochem. Biophys. Res. Commun. 225:500-504, 1996.

10. GeneBank Accession Number SP¦P80520

11. Oszvath, K. J., Hirose, H., Xia, S., and Tilson, M. D. MolecularMimicry in Human Aortic Aneurysmal Diseases. New York Academy of ScienceConference Abstract Book The Abdominal Aortic Aneurysm-GeneticPathophysiology, and Molecular Biology: 28, 1996.

12. Tanaka, S., Komori, K., Okadome, K., Sugimachi, K., and Mori, R.Detection of Active Cytomegalovirus Infection in Inflammatory AorticAneurysms with RNA Polymerase Reaction. J. Vasc. Surg. 20:235-43, 1994.

13. DePalma R. G., In R. M. Greenhalgh J. A., Mannick, and J. T. Powell(Eds). The Cause and Management of Aneurysm, London: WB SuandersCompany, 1990. Pp. 97-104.

14. IgKappa 1 is GeneBank Accession Number GI|1514581; and Ig Kappa 2 isGeneBank Accession Number GI|85921 (IgK chain v J1-region precursor)

15. “retrieve@ncbi.nlm.nih.gov

16. http://kiwi.imgen.bcm.tmc.edu:8088/bio/bio_home. html

17. Altshul S. F., Gish W., Miller W., Myers E. W., and Lipman, D. J.,Basic Local Alignment Search Tool J. Mol. Biol. 215:403-10, 1990.

18. Henikoff, S., Henikoff, J. G., Alford, W. J., and Pietrokovski, S.,Automated Construction and Graphical Presentation of Protein Blocks fromUnaligned Sequences. Gene-COMBIS, Gene. 163:GC 17-26, 1995.

19. Duret L., LALNVIEW v2.2 Available at the Biologist's Control Panelof the Human Genoma Project of the Baylor College of Medicine http://iwi.imgen.cm. mc.edu.8088/bio/bio_home. html

20. Peitsch, M. C., Promod and Swiss-Model: Internet Based Tools forAutomated Comparative Protein Modeling. Biochem Soc Trans. 24:274, 1996.Available at SwissmodΕggr.co.uk.

21. Sayle, R., Raswin Molecular Graphics, version 2.6. Freeware.

22. Korostensky C., (webmaster). AllAll.html. Available athttp://cbrg.inf.ethz.ch

23. GeneBank Accession Number SW¦P01602

24. Bork, P., Holm, L., and Sander C., The ImmunoGlobulin Fold:Structural Classification, Sequence Patterns and Common Core. J. Mol.Biol. 242:309-320, 1994.

25. Brophy, C. M., Reilly J. M., Walker-Smith G. J., Tilson, M. D. TheRole of Inflammation in Non-Specific Abdominal Aneurysm Disease. Ann.Vasc. Surg. 5:229-233, 1991.

26. Jones, E. Y., Harlos, K., Bottomley, M. J., Robinson, R. C.,Driscoll, P. C., Edwards, R. M., Clements, J. M., Dudgeon, I. J.,Stuart., D. I., Crystal structure of an Integrin-Binding Fragment ofVascular Cell Adhesion Molecular-1 at 1.8 A Resolution Stature373:539-44, 1995.

27. GeneBank Accession Number GI¦790817 (L 3486)

28. GeneBank Accession Number SP¦P19477

29. Horton, J., Ratcliffe, N. Evolution of Immunity. Immunology (4th Ed.), eds. Roitt I, Prostoff J., Male D. Mosby (Baltimore) 1996;pp15.1-15.22.

30. Xu,X., Doolittle, R. F. Presence of a Vertebrate Fibrinogen-LikeSequence in an Echinoderm. Proc. Nat. Acad. Sci. (USA) 1990;87:2097-2101.

31. Korostensky, C. (webmaster). AllAll.html. Available athttp://cbrg.inf.ethz.ch

32. Wilson, A. C., Carlson, S. S., White, T. J. Biochemical Evolution.Ann. Rev. Biochem. 1977; 46:573-639.

FOURTH SERIES OF EXPERIMENTS Experimental Details

Immunoglobulins (IgGs) purified from the aortic wall of patients withabdominal aortic aneurysms (AAAs) are immunoreactive with a human aorticprotein that we have purified and sequenced in part. (1,2,3) It hashomologies with the bovine Microfibril-Associated GlycoProtein 36 kDa(MAGP-36), which was discovered by Kobayashi et al. (4) and found tohave a tissue distribution uniquely limited to the aorta. (5) The humanprotein is named “Aortic Aneurysm Antigenic Protein 40 kDa” (AAAP-40). Asequence in AAAP-40 matches an eleven residue sequence in humanvitronectin,⁴ and a similar motif occurs in MAGP-36 (Table VIII) wAAAP-40 is immunoreactive with rabbit anti-human vitronectin antibody.(6)

Since vitronectin is synthesized in liver (not aortic adventitia), anexpression library from adventitia was screened for recombinants thatare immunoreactive with rabbit anti-human vitronectin; and the aminoacid sequences of two positive clones that strongly resemble each otherare reported. (7) This series of experiments reports the amino acidsequence of a third clone (originally the fourth clone in the series,hence “rAAAP-CL4”), because the gene product is also immunoreactive withIgG from patients with AAA.

METHODS

Human subjects: Specimens of AAA tissue and peripheral blood were takenfrom patients at the time of infrarenal AAA repair. Nocmal abdominalaorta was harvested from organ donors. The protocols for humaninvestigation have been approved by the Institutional Review Board.Surgical specimens were frozen immediately, ant then stored at −110° C.until use in biochemical studies. Serum was obtained from blood samples,an d stored at −20° C. until further studies.

Purification of IgG: Human IgG was purified separately from aortictissue and from serum as described previously. (1)

Construction of cDNA Libraries: mRNA from aortic adventitia of a AAAspecimen was reverse transcribed for insertion into the phagemid, UniZap XR™ lambda vector system, by Stratagene™ (La Jolla, Calif.). Thissystem accommodates DNA inserts up to 10 kb in length.

Expression of cDNA: The phagemid from Uni-Zap XR vector was transfectedinto a strain of E. coli (XL1-Blue MRF′, Stratagen™). The transfectedcells were plated on top agar, and then allowed to grow at 45° C. untilsmall plaques were visible (approximately 4 hours). Nitrocellulosemembranes, impregnated with 10 mm Isopropylthio-beta-D-galactopyranoside (IPTG), were placed onto the agar, andwere incubated for 4 hours at 37° C. The membranes were removed andblocked with 5% milk in TBS for 45 minutes. Incubation was continuedwith rabbit anti-human vitronectin antibody (Becton-Dickson, Bedford,Mass.) (1:10,000) for 3 hours at room temperature. After washing in TBS,the membranes were incubated with alkaline phosphatase conjugated (APC)goat anti-rabbit IgG (Sigma, St. Louis, Mo.)(1:5000) for 2 hours at roomtemperature. Following a series of washes in TBS, the membranes weredeveloped by Vectastain NBT/BICP color reagent system—(Vector,Burlingame, Calif.). The estimated frequency of the positive clones was1:10,000 on primary screening and 1:1000 on secondary screening. Thepositive plaques were rescreened to obtain pure clones.

Purification axid sequencing of DNA: Excision from positive clones wascarried out using the Ex Assist/SOLR System (Stratagene™). The phageswere collected from the agar plate using chloroform extraction, and thentransfected to SOLR cells with amplified Ex Assist Helper Phage(Stratagene™), which prevents replication of the experimental phage. Thetransformed cells were allowed to grow overnight at 37° C. onampicillin-supplemented medium which inhibits the growth ofnon-transfected cells. The cells were harvested, and then lysed byalkali buffer. DNA was purified by the phenol—chloroform extractionmethod and quantified by spectrophotometry. Agarose gel electrophoresisconfirmed the presence of plasmid DNA. DNA sequencing was carried by theDNA Sequencing Laboratory at Columbia University, New York, N.Y.

Expression of the recombinant DNA: The screened and purified plasmid wasmixed with XL1-Blue MRF′ cells pre-treated with CaCl₂. The mixture wasthen heat shocked to allow transformation. The transformed cells weregrown in non-selective media and plated onto ampicillin-containing agarto identify the plasmid-containing colonies. A single colony was grownin liquid media for 6 hours and continued to incubate for 4-6 hours with10 mM IPTG supplementation. The cells were lysed with lysozyme (Sigma,St. Louis, Mo.), and cell debris was removed by centrifugation at 20,000g for 1 hour. Protein concentration of the supernatant was determined bydye-binding assays. A non-transfected colony underwent similarprocessing to identify cellular proteins. Western immunoblots wereprobed with IgG purified from serum; of AAA patients or normalvolunteers. Alignments. The work was done with an IBM compatiblecomputer, Netscape Navigator™ 1.4, and a modem. Protein sequences weredownloaded from Retrieve at the National Institute of Health (NIH) (8)and pasted into the Pairwise Alignment Program at the Biologist'sControl Panel at the Human Genome Project of the Baylor College ofMedicine. (9) The alignments were displayed with LalnView, which is alsoavailable at the Baylor College of Medicine Web site. (9) The screen wassaved for each alignment, and then pasted into PaintBrush™ for croppingand printing. Multiple pairwise alignments were performed with MOTIF,which is also available at the Biologist's Control Panel. (9)

RESULTS

A recombinant protein was expressed by the XL1-Blue MRF′ cellstransfected with the cDNA of clone 4. Western immunoblots showed thatrAAA-CL4 was immunoreactive with IgGs-from AAA patients at a MW of 28kDa (FIG. 12). (3 out of 4 sera) It did not react with IgGs from normalvolunteers. (0 out of 3 sera)

The amino acid sequence of the protein encoded by clone 4 (rAAAP-CL4) isshown in Table IX, in alignment with several related proteins. rAAAP-CL4does not encode AAAP-40. Since the known MAGP's (MAGP-36 and MFAP-4)strongly resemble each other, and they also resemble fibrinogen-beta,(4) a pair-wise alignment of rAAAP-CL4 was carried out against thesethree proteins to evaluate their degree of relationship (Table IX).

The proteins encoded by clones 1 (rAAAP-CL1) and 5 (rAAAP-CL5) have beenreported, and these clones have lengthy sequences with high degrees ofhomology with immunoglobulins of the kappa family (IgK's). (7) They maybe matrix cellular adhesion molecules (“MAT-CAMSs”). Accordingly,alignments were done with rAAAP-CL4 against two Ig kappa sequences thatare related to clones 1 and 5 (FIG. 14).

Since the cytomegalovirus has recently been implicated as a potentialmolecular mimic in AAA disease, (10) an additional alignment ofrAAAP-CL4 was done against a cytomegalovirus protein (and against aninfluenza protein as a control).

DISCUSSION

An infiltrate of chronic inflammatory cells has been described in AAAwall. (11, 12, 13, 14) Tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and IL-8 have also been shown to be elevated in AAA tissuewhen compared to control aortic tissue. (15, 16, 17) Increases inmatrix-degrading enzymes have been found in AAA wall associated withinfiltration of mononuclear cells. (18, 19, 20) These observationssuggest that autoimmunity may play a role in the pathogenesis of AAA.

Previous work has led to the hypothesis that the self-antigens in AAAdisease belong to a family of proteins with similarities to MAGP-36. (3,7) It has also been suggested that some bacterial and viral pathogens(T. iallidum, herpes viruses, and cytomegalovirus) may be molecularmimics (like certain mycobacteria in rheumatoid arthritis). (10)Molecular mimics are pathogens with epitopes capable of initiatingautoimmunity against self-proteins. Since herpes and cytomegalovirus areso ubiquitous, it remains to explain why a relatively small number ofexposed persons eventually develop AAA. A working hypothesis is that thegenetic susceptibility resides in the MHC Class II DR-B family ofalleles. (21)

The present experiments give the deduced amino acid sequence of thefourth potential autoantigen. The experimental molecular weight ofrAAAP-CL4 (28 kDa) is not inconsistent with the deduced sequence of 193residues, since the information is incomplete in the region of the cDNAbetween the forward and reverse transcripts of the clone. The similarityof sequences from rAAAP-CL4 and the known microfibrillar proteinssuggest that it is another a member of the the MAGP family (FIG. 13);but the relationship is not as close as that of AAAP-40 to other MAGP's.The similarity of rAAAP-CL4 to the Ig kappa sequences (FIG. 14) iscompatible with the notion that it belongs to a super-family includingClones 1 and 5, which use immunoglobulin motifs in the extracellularmatrix for interaction with members of the integrin family. (22)

Finally, the resemblance of rAAAP-CL4 to a protein from cytomegalovirusis greater than that of rAAAP-CL4 to a protein from influenza (as shownin FIG. 15). This finding is consistent with the hypothesis thatcytomegalovirus might be a molecular mimic.

TABLE VIII Alignment of homologous sequences from AAAP-40, MAGP- 36,MFAP-4, and vitronectin. $ = K or R; “.” = non- conserved residue; $ F ED G V L D P D Y P AAAP-40 R F E D G V L D P D Y P VN       D G VYL . PMFAP-4       D G VYL . P MAGP-36

TABLE IX The deduced amino acid sequence of rAAAP-CL4 in single aminoacid letter code. “X” = ambiguous residue; spaces representdiscontinuities in sequence; and "--> <--“ = ambiguous region betweenforward and reverse reading frames of the nucleic acid sequence. Motifsfrom other proteins described in text are shown in alignment withrAAAP-CL4. YGLRSLSALQ XHLEACLLTS GGGXRLQEGP ATCHLPCDQA KKWNXKSQTFICMLLCPFCR 60YELRSLS         C..TT .GG      P ..C L.CD    KW......F      C.F.. MFAP-4YELK                    G..L..G           A KE.D AAAP-40YEL              LLF. G   R.Q.G     H.P.... .QW               F.R VN    SLSA.. .H   C.L.S GG       P ATC  PC.Q. Q Cytomeg XNXILKXFFFXLQFALSFPT PTTNPLFEXX XSPEPXDFQW KEKNHXGTFV SNLXFXLNIS 120.N   K.FYY SLK ALS  T P..P                    FQ.R  FV .N MFAP-4     RGFYY SLK    FP                     QW R K...G Y. ..L Y...L AAAP-40     RVYFF   QY.L..P. P   P..E.. .S..PD     EEKN              NIS VN         F .LR..LS.PT PT..V.YQ Cytomeg---------------------><-----------------------------KFF CXEFGLKAXI121-133                                                            Y.LRMFAP-4                                                     KFF    YGLRAAAP-40                                                     KYY    Y.LVN                                                       Y C.E...KA VNRSPXKAXXGM SDVIVKCPVI XFMNAKWGCK YFLNVCISNL FDXXWGFTLH PCACFPNCEE END            DV.D  PV. VF  A.W..R .F......NL            PCA MFAP-4                  P.. ..MN...G.K YY AAA-40                              C. Y....C         WG TLH ....P..EE VN                          V.WG              F...W         C.P.C Cytomeg                                                WGFT.H ..AC..N FIB-5

FOURTH SERIES OF EXPERIMENTS References

1. Gregory A K, Yin N X, Capella J, Xia S, Newman K M, Tilson M D.Feature of autoimmunity in the abdominal aortic aneurysm. Arch Surg1996: 23-25.

2. Tilson M D. Similarities of an autoantigen in aneurysmal disease ofthe human abdominal aorta to a 36-kDa microfibril-associated bovineaortic glycoprotein. Biochem. Biophys. Res. Commun. 213:40-43, 1995.

3. Xia S, Ozvath K, Hirose H, Tilson M D. Partial amino acid sequence ofa novel 40 kDa human aortic protein, with vitronectin-like,fibrinogen-like, and calcium binding domains: aortic aneurysm-associatedprotein-40 (AAAP-40) [Human MAGP-3, proposed]. Biochem Biophys ResearchCommun. 1996; 219: 36-39.

4. Kobayashi R, Mizutani A, Hidaka H. Isolation and characterization ofa 36 kDa microfibril associated glycoprotein by the newly synthesizedisoquinolinesulfonamide affinity chromatography. Bibchem Siophys Rescommun. 1994; 198:1262-6.

5. Kobayashi R, Tashima Y, Masuda H, Shozawa T, Numata Y. Miyauchi K,Hayakawa T. Isolation and characterization of a new 36-kDamicrofibril-associated glycoprotein from porcine aorta. J Biol Chem1989; 264: 17437-44.

6. Hirose H, Ozsvath K J, Xia S, Cifello V, Tilson M D. Detection ofaortic aneurysm-associated autoantigenic protein 40 kDa (AAAP-40) withantibodies to vitronectin and fibrinogen, both immunohistochemically andwith western immunoblots. Surgical Forum 1996, 82:370-373.

7. Oszvath, K J, Xia S, Hirose H, Tilson, M D. Two hypothetical proteinsof human aortic adventitia, with Ig kappa, collagenous, andaromatic-rich motifs. Biochem. Biophys. Res. Com. (1996) 225:500-504.

8. Retrieve can be accessed by e-mail at “retrievesncbi.nlm.nih.gov”

9. http://kiwi.imagen.bcmtmc.edu:8088/bio/bio_home.ht ml.

10. Ozsvath, K., Hirose, H., Xia, S., Tilson, M. D. Molecular Mimcry inHuman Aneurysmal Diseases. Annals of New York Academy of Sciences, inpress.

11. Beck, E N. Plasma Cell Infiltrates in Atherosclerotic AbdominalAortic Aneurysm. Am J Clin Path 1986; 85:21-24

12. Rizzo, R. J., McCarthy, W. J., Dixit, S. N., et al Typing ofCollagen and Content of Matrix Protein in Human Abdominal AorticAneurysm. J Vasc Surg 1989; 10:365-373

13. Koch, A. E., Haines, G. K., Rizzo, R. J. et al. Human AbdominalAortic Aneurysms: Immunophenotypic Analysis Suggesting an ImmuneMediated Response. Am J Pathol 1990; 137:1199-1219

14. Brophy, C. M. Reilly, J. M., Smith, G J W, Tilson, M. D. The Role ofInflammation in Nonspecific Abdominal Aortic Aneurysm Disease. Ann VascSurg 19091; 5:229

15. Newman, K. M. Jean-Claude, J., Li, H., Ramey, W. G., Tilson, M. D.Cytokines that Activate Proteolysis are Increased in Abdominal AorticAneurysms. Circulation 1994; 90:II224-II227

16. Szekanecz, Z., Shar M. R., Pearce, W. H. Koch, A. E. HumanAtherosclerotic Abdominal Aortic Aneurysms Produce Interleukin (IL)-6and Interferon-Gamma but not IL-2 and IL-4: The Possible Role IL-6 andInteferon-Gamma in Vascular Inflammation. Agents & Agents 1994;42:159-162.

17. Koch, A. E., Kunkel, S. L., Pearce, W. H. et al. Enhanced Productionof the Chemotactic Cytokines Interleukin-8 and Monocyte ChemoattractantProtein-1 in Human Abdominal Aortic Aneurysms. Am J Path 1993;142:I423-1431.

18. Newman, K. M., Malon, A. M., Shin, R. D., et al. MatrixMetalloproteinases in Abdominal Aortic Aneurysm: Characterization,Purification , and Their Possible Sources. Connective Tissue Res 1994;30:265-276

19. Newman, K. M., Jean-Clause, J., Li, H., Scholes, J. V., Ogata, Y.,Nagase, H., Tilson, M. D., Cellular Localization of MatrixMetalloproteinases in the Abdominal Aortic Aneurysm Wall J. Vasc Surg1994; 20:814-820.

20. Thompson, R. W. Holmes D. R., Mertens, R. A., et al. Production andLocalization of 92 Kildalton Gelatinase in Abdominal Aortic Aneurysm. AnElastolytic MetalloProteinase Expressed by Aneurysm-InfiltratingMacrophages. J. Clin Invest 1995; 96:318-326.

21. Tilson, M. D. The Genetic Susceptibility to AAA Disease Resides inMajor Histocompatibility Locus DR-Beta-1. Annals of the New York Academyof Sciences, in press.

22. Frennette, P. S., Wagner, D. D. Molecular Medicine AdhesionMolecules Part I. N. Engl J Med 1996; 334:1526-1529.

23. Genebank Accession Number: sp¦P80520

24. Genebank Accession Number: gi¦543129

25 Zhao, Z., Lee, C., Jiralerspong, S., et al. The Gene for A HumanMicrofibril-Associated Glycoprotein is Commonly Deleted in Smith MagenisSyndrome Patients. Human Mol Genetics 1995; 4:589-597.

26. Genebank Accession Number: gp¦L38486

27. Genebank Accession Number: gi¦1255044

28 Genebank Accession Number: sp¦P16748

29. Genebank Accession Number: gi¦437972

30. Genebank Accession Number: sp¦16982

5th SERIES OF EXPERIMENTS Experimental Details

The partial amino acid sequence of an aortic adventitialelastin-associated fibrillar protein may be the target of an autoimmuneresponse in patients with abdominal aortic aneurysms (AAA). (1, 2, 3)Its apparent molecular weight is approximately 40 kDa, so it has beennamed aneurysm-associated antigenic protein—40 kDa (AAAP-40). It hassimilarities to microfibril-associated glycoprotein-36 kDa (MAGP-36)(4), which was reported by Kobayashi et al. to have a tissuedistribution limited to the aorta in pig (5); unlike othermicrofibrillar proteins which appear to distribute ubiquitouslythroughout the body with elastin. (6) Accordingly, if AAAP-40 is thehuman homolog of MAGP-36, it would explain how an autoimmune reactionagainst this protein might have consequences more or less limited to theaorta and its branches.

CLONES 1 AND 5 HAVE IMMUNOGLOBULIN DOMAINS

An expression library was made from mRNA of the human aortic adventitiaand screened in an effort to clone the cDNA for AAAP-40. Two of thefirst five clones (#1 and #5) have novel features and strongly resembleeach other in domain structure. (7) While both have some features thatoccur in the families of microfibril-associated glycoproteins and othermatrix proteins (like collagen), their most remarkable property is thateach begins with a lengthy amino acid sequence (˜100 residues) that ishighly homologous to members of the immunoglobulin kappa family. Theprobability that this high degree of similarity is due to chance alone(as calculated by BlastP)(8) is in the range of 1×10⁻⁵⁰.

These are novel proteins, as matrix molecules with immunoglobulindomains have not been previously described. By analogy to themembrane-bound cell adhesion molecules (CAMs), the name Matrix CellAdhesion Molecules (MAT-CAMs) is proposed for these unique clones.Hereafter, Clone 5 will be referred to as MAT-CAM 2; since it is thesecond member of this new family to be discovered.

THREE-DIMENSIONAL MODELING OF MAT-CAM 2 (CLONE 5)

The three dimensional modeling of Clone 1 (MAT-CAM 1) is reportedelsewhere. Interestingly, the N-terminal 124 residues form a sandwich oftwo antiparallel beta sheets, characteristic of the immunoglobulinsuperfamily. The structure of the N-terminal 124 residues of MAT-CAM 2was modeled by SwissModel(9) on 11MIG. PDB, which was the most similarimmunoglobulin light chain for which a template was available (Blastpp=1.3×10⁻³¹) and displayed in three-dimensions by RasMol. (10) Theantiparallel beta sheet sandwich of MAT-CAM 2 is illustrated in FIG. 16.

FIG. 17 shows a pair-wise comparison of the modeled sequence of MAT-CAMs1 and 2 (as computed at the Biologist's Control Panel)(11) and displayedby LalnView. (12) There has obviously been substantial divergence insequence, although the structure of the immunoglobulin sandwich ispreserved. This result confirms again what has come to be seen as acommon theme in molecular evolution; namely, that structure is much morehighly conserved than sequence.

MOLECULAR EVOLUTION OF THE MAT-CAMS

The PAM distances (point accepted mutations per 100 amino acids) havebeen calculated with AllAll(13) for the MAT-CAMs, the most similarimmunoglobulins, the microfibril-associated glyco-proteins, and theirpresumed fibrinogen-like ancestor: fibrinogen-related protein-Aprecursor (SP|P19477) of the invertebrate Echinoderm sea cucumber. Forfibrinopeptide-B related proteins, a PAM unit has been estimated to beabout 1 million years. (14) FIG. 18, which is drawn roughly to scale,illustrates some of these distances. One might surmise that there was acluster of evolutionary activity among the matrix molecules around thetime of the Cambrian radiation, followed much more recently by a clusterof activity around the time of appearance of soluble immunity in thecartilagenous sharks.

Two recent findings suggest that the proteins may be authentic geneproducts. First, FIG. 19 shows the result of an immunohistochemicalexperiment which compared the binding of antibody against Ig-kappa tothe elastin-associated fibril of an aneurysmal aorta (as compared withthe binding of an antibody against Ig-heavy chain). The binding ofanti-Ig kappa to the matrix suggests that the putative MAT-CAMs areauthentic connective tissue proteins. Second, FIG. 20 illustrates anin-situ hybridization of labeled clone 1 with a section of aortic wall.The observation that binding occurs to a mesenchymal cell instead of alymphocyte buttresses the argument for authenticity.

Preliminary studies with a limited number of AAA patients and normalssuggest that MAT-CAMs 1 and 2 may also be autoantigens like AAAP-40.However, there may be a whole family of aortic antigenic proteinsrelated to the development of aneurysmal diseases.

FIFTH SERIES OF EXPERIMENTS References

1. Gregory A K, Yin N X, Capella J, Xia S, Newman K M, Tilson M D.Feature of autoimmunity in the abdominal aortic aneurysm. Arch Surg1996: 23-25.

2. Tilson M D. Similarities of an autoantigen in aneurysmal disease ofthe human abdominal aorta to a 36-kDa microfibril-associated bovineaortic glycoprotein. Biochem Biophys Res Communications 213: 40-43,1995.

3. Xia S, Ozvath K, Hirose H, Tilson M D. Partial amino acid sequence ofa novel 40 kDa human aortic protein, with vitronectin-like,fibrinogen-like, and calcium binding domains: aortic aneurysm-associatedprotein-40 (AAAP-40) [Human MAGP-3, proposed]. Biochem Biophys ResearchCommunications 1996; 219: 26-39.

4. Kobayashi R, Mizutani A, Hidaka H. Isolation and characterization ofa 36 kDa microfibril associated glycoprotein by the newly synthesizedisoquinolinesulfonamide affinity chromatography. Biochem Biophys ResCommune 1994; 198:1262-6.

5. Kobayashi R, Tashima Y, Masuda H, Shozawa T, Numata Y, Miyauchi K,Hayakawa T. Isolation and characterization of a new 36-kDamicrofibril-associated glycoprotein from porcine aorta. J Biol Chem1989; 264: 17437-44.

6. Zhao Z, Lee C-C, Jiralerspong S, Juyal R C, Lu F, Baldini A,Greenberg F, Caskey C T, Patel P I. The gene for a humanmicrofibril-associated glycoprotein is commonly deleted in Smith-Magenissyndrome patients. Human Mol Genetics 1995; 4: 589-597.

7. Ozsvath K J, Xia Shichao, Hirose H, Tilson M D. Two hypotheticalproteins of human aortic adventitia, with Ig Kappa, collagenous, andaromatic-rich motifs. Biochem Biophys Research Communications (1996)225: 500-504.

8. Altschul S F, Gish W, Miller W. Myers E W, Lipman D J. Basic localalignment search tool. J Mol Biol 1990; 215: 403-10.

9. Peitsch M C. Promod and swiss-model: internet based tools forautomated comparative protein modeling. Biochem Soc Trans 1996; 24: 274.Available at Swissmod@ggr.co.uk.

10. Sayle R. RasWin Molecular Graphics, version 2.6. Freeware.

11. http://kiwi.imgen.bcm.tmc.edu:8088/bio/bio_home.html

12. Duret L. LALNVIEW v 2.2, available at the Biologist's Control Panel(reference above).

13. Korostensky C (webmaster). AllAll. html, available athttp://cbrg.inf.ethz.ch.

14. Wilson A C, Carlson S S, White T J. Biochemical evolution. Ann RevBiochem 1977; 46:573-639.

SIXTH SERIES OF EXPERIMENTS Experimental Details

Aortic aneurysms are a common disease of maturity, among the ten leadingcauses of death in men and women of ages 55-74. (1) Despite themortality and suffering caused by this problem, research into the causesof the abdominal aortic aneurysm (AAA) has been neglected. According toa recent search of the computer resources at the National Institutes ofHealth (NIH) on the subject of funded grants, the NIH is spendingsomething like 1.7 billion dollars a year on AIDS, compared to a fewhundred thousand dollars on aneurysms; even though almost as many peopledie each year from aortic aneurysms as from AIDS (about 25,000versus′34,000). (1)

Autoimmunity has been implicated in the pathogenesis of the AAA.Immunoglobulin-G (IgG) purified from the wall of AAA specimens isimmunoreactive with proteins of ˜80 kDa and ˜40 kDa extractable from theaortic matrix and with matrix elements that resemble elastin-associatedmicrofibrils in immunohistochemical preparations. (2,3) This putativeautoantigen has been purified partially sequenced. (4)

The notion that aneurysmal disease has features in common withautoimmune diseases like rheumatoid arthritis opens the way for many newresearch approaches to the issues of treatment and prevention. Ifspecific antibodies are detectable in serum, as preliminary resultssuggest, it may be possible to detect susceptibility to the diseasebefore significant aortic degeneration has occurred. If tolerance forthe aortic autoantigen can be induced, it may be possible todown-modulate progression of aortic degeneration (as has beenaccomplished in rheumatoid arthritis (5)).

Clifton (6) reported an instance of familial clustering of AAA and 15Tilson (7,8) postulated a general genetic basis for susceptibility.Numerous subsequent studies have added support to the genetichypothesis. (9-21) The most recent statistical model for the mode ofinheritance predicts a dominant gene. (22)

Three candidate genes have been proposed. 1) A mutated gene for thealpha chain of Type III Collagen (COL3A1) was found to cosegregate withaneurysmal disease in one family,(23) but further studies revealed onlytwo additional positive families in another 50 evaluated. (24) 2) Adeficiency allele for alpha-1 antitrypsin (MZ) was found in five (11%)of 47 patients, (25) so if this gene has etiological significance insome individuals, it accounts for a relatively small subset. 3) Anidentical nucleotide substitution in the cDNA for Tissue Inhibitor ofMetalloproteinases (TIMP) was found in two of six patients;(26) but thisfinding turned out to be trivial, since the polymorphism occured in thethird position of the codon. Accordingly, the gene and/or genesresponsible for most cases of AAA remain to be discovered.

Evidence for Autoimmunity in the AAA

The walls of abdominal aortic aneurysms (AAA's) contain abundant cellsof chronic inflammation, particularly in the adventitia. (27-29) Russellbodies have also been observed in the AAA tissues, which are a featureof autoimmune diseases like Hashimoto's thyroiditis. (28) Additionalfindings from since that time include 1) elevated levels of cytokinesthat activate proteolysis in AAA tissue; (30) 2) elevated content ofplasmin (31) (the principal activator of the matrixmetallo-proteinases); 3) increased amounts of proteins immunoreactivewith antibodies against collagenase (MMP-1)(32), stromelysin (MMP-3),and gelatinase-B (MMP-9). (33) In our original study of 1991 we notedsubstantial increases in the IgG present in the walls of aorticaneurysmal tissue by comparison to atherosclerotic occlusive disease andcontrol aortas (28). This observation led to further work to identifythe putative autoantigen.

Purification of an Autoantigen

IgG from aneurysm wall by affinity to Protein-A was purified and used asa probe in immunohistochemical preparations of normal aorta to determinewhether the autoantigen is an aortic matrix protein. (2) Taking asimilar approach to the assignment of a molecular weight to the putativeautoantigen, used the AAA IgG was used as a probe on Western immunoblotsof soluble proteins extracted from the normal aorta. (2)

Amino acid sequencing of peptides from the 80 kDa immunoreactive protein(3) led to a postulation that the autoantigen of AAA disease is thehuman homolog of a bovine aortic protein discovered by Kobayashi, namedmicrofibril-associated glycoprotein-36 (MAGP-36). (34) MAGP-36 occurs innature as a dimer, so it was hypothesized that the 80 kDa protein isalso a dimer. Further extractions under chaotropic reducing conditionswere used to purify sufficient human protein with Mr ˜40 kDa to 1)confirm that the 40 kDa species is also immunoreactive with AAA IgG; and2) confirm that it is closely related to the MAGP faiqily by directamino acid sequencing. (4)

At first this 40 kDa protein was called “Aortic Aneurysm AntigenicProtein-40” (AAAP-40). (4) Since it appears to be the third member ofthe MAGP family to be discovered in man, the abbreviation MAGP-3 may bepreferred in the future. Tables I and II (in the First Series ofExperiments) display similarities of AAAP-40 with two otherwell-characterized microfibril-associated glycoproteins: MAGP-36(34) andMFAP-4. (35)

Fibrinogen-like domains are well-known in the MAGP's. The sequence ofAAA-40 shown in Table I (in the First Series of Experiments) has regionsof homology with sequences in the alpha and beta chains of fibrinogen.Another sequence that we have determined (data not shown) matchesresidues 283-292 in the gamma chain. Since the three fibrinogen chainsare believed to have a single ancestral gene, it would appear likelythat AAAP-40 is related to the common ancestor since it has motifs thatare used in all three fibrinogen subunits.

Kobayashi et al noted that MAGP-36 has the property of calcium-binding,(34) although a candidate site for the calcium-binding domain has notbeen proposed. Kielty and Shuttleworth have observed that incubation ofintact microfibrils with EDTA rapidly results in gross disruption ofmicrofibrillar organization, which can be reversed by replacing calcium.(36) Since fibrillin has 43-EGF-like motifs with calcium bindingconsensus sequences, and calcium has been proposed to orchestrate theassembly of tropoelastin to the microfibril and hold it in register forcrosslinking,(37) it was hypothesize that the calcium-binding domain ofAAAP-40 may play a role in calcium-dependent microfibril assembly in theaorta. When GenBank was searched for homologies of AAAP-40 and MAGP-36,sequences were found in calcium-binding myeloid-related protein(>pir¦A44111: #144-154), the calcium-binding domain of humanfibrinogen-beta, and bovine aggrecan (>pir¦A39808: #59-66) that havesimilarities to MFAP-4, MAGP-36, and AAAP-40. Bold type is used in TableII (in the First Series of Experiments) to highlight residues thatappear to be conserved, with possible significance for thecalcium-binding function.

Another matrix protein detected in human embryonic tissue (sulfatedprotein 30 kDa=SP-30) has been reported to be immunoreactive withmonoclonal antibodies against human vitronectin. (38) A sequence ofAAAP-40 that matches residues #230-240 in human vitronectin is shown inTable II (in the First Series of Experiments). Tomasini-Johansson et alproposed that SP30 is the human homolog of MAGP-31, but since MAGP-31does not have this twelve residue vitronectin-like domain, it isbelieved that SP-30 is more likely to be closely related to AAAP-40, orto another MAGP with a vitronectin-like domain that is aubiquitouslyexpressed during embryogenesis.

Parallels With Rheumatoid Arthritis

The association of rheumatoid arthritis (RA) and Class II MajorHistocompatibility Locus (MHC) DR4 was first reported by Stastny in1978. (39) However, it became evident that DR4 was not always associatedwith RA in different racial/ethnic populations, and the reader isreferred to a review article by Ollier and Thomson (40) which describesthe state of the field leading to the formulation of the hypothesis ofthe shared epitope by Gregersen, Silver, and Winchester in 1987. (41)The hypothesis is that arthritogenic DR molecules share a highlyconserved sequence of amino acids in their third hypervariable region(amino acid positions 70-74).

In the context of a rather homogeneous population seen by the arthritisgroup at the Mayo Clinic, evidence for the shared epitope seemscompelling. Weynand and colleagues have reported that 98 of 102 (96w)patients express one of the major North American disease-linkedpolymorphisms (*04, *0101, or *1402). (42) Forty-seven patients carrieda double dose of the relevant sequence stretch. Nodular disease wasexpressed in 100% of the patients typed as HLA-DRB1*04/04. Patients witha double dose of the shared sequence tend to have more severemanifestations of rheumatoid disease.

A Pilot Study to Test the Notion of an Association with MHC Class II DR

As a pilot study we 26 patients have been tissue typed with AAA's.Because of the demographics of the patient population of our hospitalsystem, there were five Americans of color in the sample. Since AAA'sare much less common in African Americans than in Caucasians, these tenhaplotypes are particularly interesting. It has been a workinghypothesis of Tilson that Americans of color might have a double dose ofthe susceptibility allele. The results of allele frequency analysis inthese five patients compared with the expected frequencies are presentedin Table X. The expected frequencies were derived from the tablespublished by the Eleventh International Histocompatibility Workshop heldin Yokohama, Japan in November 1991. (43) Only three DR alleles weredetected among the ten haplotypes of interest. The data in Table Xsuggest that patients with alleles for DR 2, 12 and 13 occurredsignificantly more often than expected by chance alone (p=3×10⁻⁴).

Inspection of the amino acid sequences of the most common alleles forthese. three DRB1 types revealed that the residues that they have incommon are at positions 31 and 47 of the second hypervariable region;specifically, both residues are phenylalanines (phe). (44) The otheralleles that have phe at positions 31 and 47 are DR 3 and DR 11.Revisiting the data on the 21 non-Black patients in the original series,16 (75%) have an allele with phenylalanine at positions 31 and 47; andfour of these have a double dose of one of the putative “aneurysmogenic”alleles.

If the requirement for a phe at position 47 is relaxed to permit a pheat position 37, one hundred percent (21 out of 21) non-Black patientshave a putative aneurysmogenic allele; and 50% of the total patients(13/26) have a double dose. Thus, there is now a testable hypothesis toevaluate going forward to look at additional patients prospectively.

The notion that AAA is an autoimmune disease of maturity like rheumatoidarthritis offers many opportunities for new knowledge, including 1) abetter understanding of etiological influences; 2) novel diagnosticapproaches to the detection of susceptibility (DR) and disease activity(serum IgG level) before the aorta has commenced to dilate; and, 3)therapeutic interventions (induction of tolerance) that might change thenatural history of the disease. In terms of etiology, several instancesof molecular mimicry by common (and uncommon) pathogens have been found,(45) wherein the shared epitopes may provide informative explanationsnot only for features of AAA disease, but also for previouslyunexplained phenomena related to the clinical manifestations of certaininfections.

TABLE X Allele frequencies in five Americans of Color with AAA (AOC-AAA,ten haplotypes) with the expected frequencies for these alleles in NorthAmerican Blacks (NAB) as reported by the International Workshop in 1991(Table 12, 132 haplotypes).⁴³ The cumulative probability of this resultis p = 3 × 10⁻⁴. Probabilities were calculated by Fisher's Exact Testfor 2 × 2 Contingeny Tables. Allele Frequency in AOC-AAA Frequency inNAB p DRB1*02 40% 12% .037 DRB1*12 30%  5% .023 DRB1*13 30% 15% .370

SIXTH SERIES OF EXPERIMENTS References

1. Silverberg E, Lubera J A. Cancer statistics. CA—A Cancer Journal forClinicians 1996; 46: 5-27.

2. Gregory A K, Yin N X, Capella J, Xia S, Newman K M, Tilson M D.Features of autoimmunity in the abdominal aortic aneurysm. Archives ofSurgery, 1996; 131: 85-88.

3. Tilson M D. Similarities of an autoantigen in aneurysmal disease ofthe human abdominal aorta to a 36-kDa microfibril-associated bovineaortic glycoprotein. Biochem Biophys Res Communications 213: 40-43,1995.

4. Xia S, Ozsvath K, Hirose H, Tilson M D. Partial amino acid sequenceof a novel 40 kDa human aortic protein, with vitronectin-like,fibrinogen-like, and calcium binding domains: aortic aneurysm-associatedprotein-40 (AAAP-40) [Human MAGP-3, proposed]. Biochem Biophys ResearchCommunications 1996; 219: 36-39.

5. Trentham D E, Dynesium-Trentham R A, Oran E J, et al. Effects of oraladministration of type II collagen on rheumatoid arthritis. Science.1993; 261: 1727-1730.

6. Clifton M. Familial abdominal aortic aneurysms. Br J Surg 1977;64:765-766.

7. Tilson, M D, in discussion of Busuttil RW, Abou-Zamzam A M, MachlederH I. Collagenase activity of the human aorta: A comparison of patientswith and without abdominal aortic aneurysms. Arch Surg 1980; 115:1373-8.

8. Tilson M D, Dang C. Generalized arteriomegaly—a possiblepredisposition to the formation of abdominal aortic aneurysms. Arch Surg1981; 16:1030.

9. Tilson M D, Seashore M R. Human genetics of abdominal aorticaneurysm. Surg Gynecol Obstet 1984; 15:129.

10. Tilson M D, Seashore M R. Fifty families with abdominal aorticaneurysms in two or more first-order relatives. Am J Surg 1984; 147:551-553.

11. Tilson M D, and Seashore M R. Ninety-four families with clusteringof abdominal aortic aneurysms (AAA) Circulation, Part II 1984; 70:II-141.

12. Norrgard O, Rais O, Angquist K A. Familial occurrence of abdominalaortic aneurysms. Surgery 1984;95:650-656.

13. Johansen K, Koepsell T. Familial tendency for abdominal aorticaneurysms. JAMA 1986; 256: 1934-1936.

14. Powell J T, Greenhalgh R M. Multifactorial inheritance of abdominalaortic aneurysm. Eur J Vasc Surg 1987; 1:29-31.

15. Cole C W, Barber G G, Bouchard A G, McPhail N V, Roberge C, WaddellW G, Wellington J L. Abdominal aortic aneurysm: Consequences of apositive family history. Can J Surg 1988;32:117-120.

16. Collin J, Walton J. Is abdominal aortic aneurysm a familial disease?Br Med J 1989; 299: 493.

17. Darling III R C, Brewster D C, Darling R C, et al. Are familialabdominal aortic aneurysms different? J Vasc Surg 1989; 10:39-43.

18. Loosemore T M, Child A H, Dormandy J A. Familial abdominal aorticaneurysms. J Roy Soc Med 1988; 81: 472-3.

19. Bengtsson H, Norrgard 0, Angquist K A, Eckberg O, Obereg L,Bergqvist D. Ultrasonographic screening of the abdominal aorta amongsiblings of patients with abdominal aortic aneurysms. Br J Surg 1989;76: 589-591.

20. Webster M W, St. Jean P L, Steed D L, et al. Abdominal aortiCaneurysm: Results of a family study. J Vasc Surg 1991; 13:366-372.

21. Majumder P P, St. Jean P L, Ferrell R E, Webster M W, Steed D L. Onthe inheritance of abdominal aortic aneurysm. Am J Hum Genet 1991; 48:164-170.

22. Verloes A, Sakalihasan N, Koulischer L, Limet R. Aneurysms of theabdominal aorta: familial and genetic aspects in three hundredpedigrees. J Vasc Surg 1995; 21: 646-55.

23. Kontusaari S, Tromp G, Kuivaniemi H, Romanic A, Prockop D J. Amutation in the gene for Type III procollagen (COL3A1) in a family withaortic aneurysms. J Clin Invest-1990; 86:1465-1473.

24. Tromp G, WU Y, Prockop D J, et al (24 authors). Sequencing of cDNAfrom 50 unrelated patients reveals that mutations in the triple-helicaldomain of Type III procollagen are an infrequent cause of aorticaneurysms. J Clin Invest. 1993; 91:2539-2545.

25. Cohen J R, Sarfati I, Ratner L, Tilson M D. Alpha-1 antitrypsinphenotypes in patients with abdominal aortic aneurysms. J Surg Res 1991;49:319-321.

26. Tilson M D, Reilly J M, Brophy C M, Webster E L, Barnett T R.Expression and sequence of the gene for tissue inhibitor ofmetalloproteinases in patients with abdominal aortic aneurysms. J VascSurg 1993; 18: 266-70.

27. Koch A E, Haines G K, Rizzo R J, Radsevich J A, Pope R M, Robinson PG, Pearce W H. Human abdominal aortic aneurysms: Immunophenotypicanalysis suggesting an immune mediated response. Am. J. Pathol 1990;137: 1199-1219.

28. Brophy C M, Reilly J M, Walker-Smith G J, Tilson M D. The role ofinflammation in non-specific abdominal aortic aneurysm disease. Annalsof Vascular Surgery 1991; 5: 229-233.

29. Beckman E N. Plasma cell infiltrates in abdominal aortic aneurysms.Amer. J. Clin. Path. 1986; 85: 21-24.

30. Newman K M, Jean-Claude J, Li H, Ramey W G, Tilson M D. Cytokinesthat active proteolysis are increased in abdominal aortic aneurysms.Circ. 1994; 90: 224-227.

31. Jean-Claude J, Newman K M, Li H, Tilson M D. Possible key role forplasmin in the pathogenesis of abdominal aortic aneurysms. Surgery,1994; 116:472-8.

32. Irizarry E, Newman K, Gandhi R, Nackman G, Wishner S, Tilson M D.Demonstration of interstitial collagenase in abdominal aortic aneurysms.J Surg Res 1993; 54: 571-574.

33. Newman K M, Ogata Y, Malon A M, Irizarry E, Gandhi R H, Nagase H,Tilson M D. Identification of matrix metalloproteinases 3(stromelysin-1) and 9 (gelatinase B) in abdominal aortic aneurysm.Arteriosclerosis and Thrombosis, 1994; 14: 1315-1320.

34. Kobayashi R, Mizutani A, Hidaka H. Isolation and characterization ofa 36-kDa microfibril-associated glycoprotein by the newly synthesizedisoquinolinesulfonamide affinity chromatography. Biochem Biophys ResCommunic 1994; 198: 1262-6.

35. Zhao Z, Lee C-C, Jiralerspong S, Juyal R C, Lu F, Baldini A,Greenberg F, Caskey C T, Patel P I. The gene for a humanmicrofibril-associated glycoprotein is commonly deleted in Smith-Magenissyndrome patients. Human Mol Genetics 1995; 4: 589-597.

36. Kielty C M, Shuttleworth C A (1993). The role of calcium in theorganization of the fibrillin microfibrils. FEBS 336: 323-326.

37. Mecham R P, Heuser J E (1991). in Cell Biology of ExtracellularMatrix (Hay ED, ed) 2nd Ed, pp 79-109, Plenum Press, New York.

38. Tomasini-Johansson B R, Ruoslahti E, Pierschbacher M D (1993). A 30kDa sulfated extracellular matrix protein immunologically crossreactivewith vitronectin. Matrix 13: 203-214.

39. Stastny P. Association of the B-cell alloantigen DRw4 withrheumatoid arthritis. N Engl J Med 1978; 298: 869-871.

40. Ollier W, Thomson. Population genetics of rheumatoid arthritis.Rheum Dis Clinics of North Amer 1992; 18: 741-759.

41. Gregersen P K, Silver J, Winchester R J. The shared epitopehypothesis: an approach to understanding the molecular genetics ofsusceptibility to rheumatoid arthritis. Arthritis Rheum 1987; 30:1205-13.

42. Weynand C M, Hicok K C, Conn D L, Goronzy J J. The influence ofHLA-DRB1 genes on disease severity in rheumatoid arthritis. Ann Int Med1992; 117: 801-6.

43. Tsuji K, Aizawa M, Sasazuki T. HLA 1991: Proceedings of the EleventhInternational Histocompatibility Workshop and Conference held inYokohama, Japan, 6-13 November 1991. Oxford Univ Press, Oxford, 1992; 1:Table 12: W15.1.

44. Lechler R I, Simpson E, Bach F H. Major and Minor HistocompatibilityAntigens: An Introduction. in Transplantation Immunology, eds. FH Bach,H. Auchincloss Jr. Wiley-Liss, New York, 1995, pp 1-34.

45. Ozsvath K J, Hirose H, Xia S, Tilson M D. Molecular mimicry in humanaortic aneurysmal diseases (abstract). Annals of the NY Acad Sciences,in press.

46. GenBank accession number >pir¦A44111: residues 144-154.

47. GenBank accession number >pir¦A39808: residues 59-66.

SEVENTH SERIES OF EXPERIMENTS Experimental Details

Autoimmunity has been implicated in the pathogenesis of the abdominalaortic aneurysm (AAA). (1-3) Studies to identify one of the putativeautoantigens have revealed amino acid sequences homologous to the bovineaortic microfibril-associated glyprotein-36 kDa (MAGP-36). (4,5) We havepartially sequenced this putative autoantigen, (6) and computerizedsearches have revealed homologies with proteins of two microorganismsassociated with aneurysmal conditions: Treponema pallidum (T pall) andherpes simplex. The concept of molecular mimicry suggests that aneurysmsin these infectious conditions might be on the basis of shared epitopes.In other words, an immune response against the pathogen might alsoattack an essential structural protein of the aortic matrix. The presentexperiments were carried out to test the hypothesis that the aorticprotein shares epitopes with these pathogens.

METHODS

Immunohistochemistry was performed using rabbit anti-T pall and rabbitantiherpes antibodies (ViroStat, Portland, Me.) versus sections ofnormal and aneurysmal aorta.

Purified proteins of T pall (T pure, Lee Laboratories, Grayson, Ga.)were separated by SDS-PAGE electrophoresis (12. 50%), and immunoblotswere prepared with the above rabbit antibodies and also with purifiedIgGs obtained from AAA wall and control serum. The filters were blockedwith 5% dry milk in PBS prior to incubating with first antibody. After aseries of washes, the filters were incubated with a second antibody;alkaline phosphatase-conjugated antibody (goat anti-human IgG, Sigma,St. Louis, Mo., for the filters incubated with human IgG, 1:1000, andgoat anti-rabbit IgG for the filter incubated with anti-T pall, 1:2500).

The filters were developed using Vectastain NBT-BICP (VectorLaboratories, Burlingame, Calif.).

RESULTS

Rabbit antibodies against T pall and herpes bound to the adventitialelastin-associated microfibril (FIG. 21). Immunoreactive proteins in Tpall were also identified with both rabbit antibodies, and also with IgGpurified from AAA wall (FIG. 22).

DISCUSSION

The notion of molecular mimicry is that epitopes of microbial pathogensmay so closely resemble some of the host's normal proteins thatinfection with the organism may trigger an autoimmune disease. (7)Herpes simplex virus has recently been implicated as possibly havingsome role in the abdominal aortic aneurysm. (8) Considering the presenthypothesis that herpes may exert its influence on the pathogenesis ofthe aortic aneurysm by molecular mimicry, the observations of DePalmaabout an outbreak of ruptured aneurysms in a colony of Capuchin monkeysare quite extraordinary. (9) Several years prior to the manifestationsof aneurysmal disease in the monkeys, they had been experimentallyinfected at the National Institutes of Health with herpes virus.

Similarly, the relationship between the development of luetic aneurysmsin patients infected with T pall has never been adequately explained.The usual explanation that aortic smooth muscle cells die ofmalnutrition because the spirochetes proliferate in the vasa vasorumseems far-fetched. (9) It is hypothesized that an epitope is shared bythe spirochete and the elastin-associated aortic microfibril, whichtriggers the destruction of aortic matrix.

In summary, it is hypothesized that herpes and syphilis are associatedwith auto-immune destruction of structural load-bearing elements of theaorta because of epitopes shared with the aortic elastin-associatedmicrofibril. Recent assignment of the gene responsible for Marfansyndrome to fibrillin, which is a component of the microfibril,underscores the importance of microfibrillar integrity in preventinganeurysmal dilation.

The present finding have diagnostic and therapeutic implications. Fromthe diagnostic perspective, it may be possible to develop a serum testfor immune titer against the autoantigen, which might identifyindividuals susceptible to aneurysm formation prior to aorticenlargement. From the therapeutic perspective, it may be possible in thefuture to induce tolerance to the autoantigen in the AAA-susceptibleindividual, possibly slowing or preventing progression of the disease.

These findings suggest that the basis for the association of lues andherpes with aneurysmal diseases may be due to molecular mimicry of thesepathogens with epitopes of the normal human aortic elastin-associatedmicrofibril.

REFERENCES FOR SEVENTH SERIES OF EXPERIMENTS

1. Koch, A. E., G. K. Haines, R. J. Rizzo, J. A. Radsevich, R. M. Pope,P. G. Robinson & W. H. Pearch. 1990. Human abdominal aortic aneurysms:Immunophenotypic analysis suggesting an immune mediated response. Am. J.Pathol. 137:1199-1219.

2. Brophy, C. M., J. M. Reilly, G. J. Walker-Smith & M. D. Tilson. 1991.The role of inflammation in non-specific abdominal aortic aneurysmdisease. Ann. Vasc. Surg. 5:229-233.

3. Gregory, A. K., N. X. Yin, J. Capella, S. Xia, K. M. Newman & M. D.Tilson. 1996. Features of autoimmunity in the abdominal aortic aneurysm.Arch. Surg. 131:85-88.

4. Tilson, M. D. 1995. Similarities of an autoantigen in aneurysmaldisease of the human abdominal aorta to a 36-dDa microfibril-associatedbovine aortic glycoprotein. Bio-chem. Biophys. Res. Commun. 213:40-43.

5. Kobayashi, R., A. Mizutani & H. Hidaka. 1994. Isolation andcharacterization of a 36-kDa microfibril-associated glycoprotein by thenewly synthesized isoquinoline-sulfonamide affinity chromatography.Biochem. Biophys. Res. Commun. 198:1262-1266.

6. Xia, S., K. Ozsvath, H. Hirose & M. D. Tilson. 1996. Partial aminoacid sequence of a novel 40 kDa human aortic protein, withvitronectin-like, fibrinogen-like, and calcium binding domains: Aorticaneurysm associated protein −40 (AAAP-40) [Human MAG-3, proposed].Biochem. Biophys. Res. Commun. 219:36-39.

7. Fujinami, R. S. & M. B. Oldston. 1989. Molecular mimicry as amechanism for virus-induced autoimmunity. Immunol. Res. 8:3-15.

8. Tanaka, S., K. Komori, K. Oradome, K. Sugimachi & R. Mori. 1994.Detection of active cytomegalovirus infection in inflammatory aorticaneurysms with RNA polymerase chain reaction. J. Vasc. Surg. 20:235-243.

9. DePalma, R. G. 1990. In The Cause and Management of Aneurysma. R. M.Greenhalgh, J. A. Mannick & J. T. Powell, Eds.: 97-104. W. B. SaundersCompany. London.

10. Dietz, H. C., G. R. Cutting, R. E. Pyeritz, et al. 1991. Marfansyndrome caused by a recurrent de novo missense mutation in thefibrillin gene. Nature 352:337-339.

57 1 105 PRT Homo sapiens Xaa=unknown amino acid 1 Asn Glu Asn Asn ValVal Asn Glu Tyr Ser Gln Glu Leu Glu Lys Xaa 1 5 10 15 Phe Glu Asp GlyVal Leu Asp Pro Asp Tyr Pro Xaa Trp Thr Val Phe 20 25 30 Gln Xaa Tyr PhePro Phe Val Asp Leu Met Val Met Ala Asn Gln Pro 35 40 45 Met Gly Glu LysTyr Tyr Asp Phe Phe Gln Tyr Thr Xaa Gly Met Ala 50 55 60 Lys Glu Tyr AspGly Phe Gln Tyr Thr Xaa Gly Met Ala Lys Xaa Ala 65 70 75 80 Gly Asn AlaLeu Met Asp Gly Ala Ser Gly Leu Met Xaa Trp Xaa Gln 85 90 95 Trp Arg GlyPhe Tyr Tyr Ser Leu Lys 100 105 2 307 PRT Artificial SequenceDescription of Artificial Sequence Source Synthesized 2 Met Asp Met ArgVal Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro GlyAla Arg Cys Asp Ile Gln Leu Thr Gln Ser Pro Ser Leu 20 25 30 Leu Ser AlaSer Val Gly Asp Arg Val Met Ile Thr Cys Arg Ala Ser 35 40 45 Gln Ala IleSer Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro LysLeu Leu Ile His Ala Ala Ser Ser Leu Gln Thr Gly Val 65 70 75 80 Pro SerArg Phe Ser Gly Ser Gly Ser Gly Thr Xaa Phe Thr Leu Thr 85 90 95 Ile SerXaa Leu Gln Ser Glu Xaa Leu Gln Leu Tyr Tyr Cys Gln His 100 105 110 LeuLys Gly Tyr Pro Ile Thr Phe Arg Pro Arg Asp Thr Xaa Gly Xaa 115 120 125Xaa Xaa Asn Cys Xaa Cys Thr Ile Xaa Ser Ser Ser Ser Arg His Leu 130 135140 Xaa Asn Ile Glu Ile Trp Xaa Cys Leu Cys Cys Xaa Ala Cys Xaa Ile 145150 155 160 Thr Ser Xaa Pro Lys Lys Ala Lys Phe His Trp Lys Val Asp AsnPro 165 170 175 Ser Asn Arg Val Thr Pro Gln Lys Asn Phe Pro Xaa Gln LysVal Phe 180 185 190 Glu Asn Phe Gly Gln Gly Lys Xaa Xaa Pro Gly Gly AlaLys Val Gln 195 200 205 Gly Glu Gly Gly Lys Xaa Leu Pro Ile Gly Xaa PhePro Xaa Glu Cys 210 215 220 Xaa Gln Ser Xaa Thr Ala Arg Thr Ala Leu ThrAla Ser Ala Ala Pro 225 230 235 240 Thr Arg Lys His Lys Val Tyr Ala LysGlu Val Thr His Gln Gly Leu 245 250 255 Xaa Ser Ser Ser Leu Thr Pro SerHis Pro Leu Ala Xaa Xaa Asp Pro 260 265 270 Phe Ser Thr Gly Asp Leu ProLeu Leu Arg Ser Ser Ser Xaa Phe Phe 275 280 285 Thr Ser Pro Pro Ser SerSer Leu Ala Leu Ile Met Leu Met Leu Glu 290 295 300 Glu Asn Glu 305 3293 PRT Artificial Sequence Description of Artificial Sequence SourceSynthesized 3 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu TrpIle Ser 1 5 10 15 Gly Ala Asn Gly Asp Ile Val Met Thr Gln Ser Pro AspSer Leu Gly 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg SerSer Gln Arg 35 40 45 Leu Phe Phe Gly Ser Asn Ser Lys Asn Tyr Leu Ala TrpTyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp AlaSer Thr Arg 65 70 75 80 Asp Ser Gly Val Leu Thr Asp Ser Leu Ala Ala GlyLeu Gly Xaa Ile 85 90 95 Ser Leu Ser Pro Ser Xaa Xaa Cys Arg Leu Lys AsnLeu Ile Leu Xaa 100 105 110 Leu Ser Ala Ile Ile Ile Ile Ser Xaa Xaa ThrPhe Arg Pro Trp Gly 115 120 125 Thr Xaa Leu Xaa Ile Gln Xaa Lys Cys TrpXaa Ala Xaa Ile Phe Xaa 130 135 140 Ser Phe Phe Pro Pro Xaa Glu Lys GlnPhe Lys Xaa Xaa Xaa Phe Phe 145 150 155 160 Phe Phe Ser Pro Phe Leu XaaGly Trp Xaa Leu Gly Xaa Leu Phe Xaa 165 170 175 Gly Pro Xaa Glu Lys IlePhe Phe Pro Xaa Gly Pro Lys Lys Arg Gly 180 185 190 Arg Gly Xaa Lys XaaPro Pro Asn Trp Gly Lys Ser Pro Ser Gly Xaa 195 200 205 Xaa Xaa Gly ArgGly Xaa Gln Gly Lys Gly Asn Leu Lys Ala Leu Trp 210 215 220 Xaa Glu ProXaa Arg Leu Gly Lys Gly Gly Ile Arg Gly Xaa Asn Lys 225 230 235 240 XaaXaa Ala Xaa Glu Val Thr His Ser Gly Leu Ser Phe Ala Xaa Ser 245 250 255Lys Lys Xaa Xaa Gln Gly Arg Xaa Leu Glu Gly Glu Val Pro Pro Pro 260 265270 Val Xaa Xaa Xaa Gln Pro Asp Pro Leu Pro Ser Phe Gly Leu Xaa Pro 275280 285 Phe Phe His Arg Gly 290 4 196 PRT Artificial SequenceDescription of Artificial Sequence Source Synthesized 4 Tyr Gly Leu ArgSer Leu Ser Ala Leu Gln Xaa His Leu Glu Ala Cys 1 5 10 15 Leu Leu ThrSer Gly Gly Gly Xaa Arg Leu Gln Glu Gly Pro Ala Thr 20 25 30 Cys His LeuPro Cys Asp Gln Ala Lys Lys Trp Asn Xaa Lys Ser Gln 35 40 45 Thr Phe IleCys Met Leu Leu Cys Pro Phe Cys Arg Xaa Asn Xaa Ile 50 55 60 Leu Lys XaaPhe Phe Phe Xaa Leu Gln Phe Ala Leu Ser Phe Pro Thr 65 70 75 80 Pro ThrThr Asn Pro Leu Phe Glu Xaa Xaa Xaa Ser Pro Glu Pro Xaa 85 90 95 Asp PheGln Trp Lys Glu Lys Asn His Xaa Gly Thr Phe Val Ser Asn 100 105 110 LeuXaa Phe Xaa Leu Asn Ile Ser Lys Phe Phe Cys Xaa Glu Phe Gly 115 120 125Leu Lys Ala Xaa Ile Arg Ser Pro Xaa Lys Ala Xaa Xaa Gly Met Ser 130 135140 Asp Val Ile Val Lys Cys Pro Val Ile Xaa Phe Met Asn Ala Lys Trp 145150 155 160 Gly Cys Lys Tyr Phe Leu Asn Val Cys Ile Ser Asn Leu Phe AspXaa 165 170 175 Xaa Trp Gly Phe Thr Leu His Pro Cys Ala Cys Phe Pro AsnCys Glu 180 185 190 Glu Glu Asn Asp 195 5 58 PRT Homo sapiensXaa=unknown amino acid 5 Tyr Phe Pro Phe Val Asp Leu Met Val Met Ala AsnGln Pro Met Gly 1 5 10 15 Glu Tyr Tyr Asp Phe Phe Gln Tyr Thr Xaa GlyMet Ala Lys Glu Tyr 20 25 30 Asp Gly Phe Gln Tyr Thr Xaa Gly Met Ala LysIle Tyr Ala Gly Asn 35 40 45 Ala Leu Met Asp Gly Ala Ser Gly Leu Met 5055 6 60 PRT Homo sapiens 6 Thr Leu Lys Gln Lys Tyr Glu Leu Arg Val AspLeu Glu Asp Phe Glu 1 5 10 15 Asn Asn Thr Ala Tyr Ala Lys Tyr Ala AspPhe Ser Ile Ser Pro Asn 20 25 30 Ala Val Ser Ala Glu Glu Asp Gly Tyr ThrLeu Phe Val Ala Gly Phe 35 40 45 Glu Asp Gly Gly Ala Gly Asp Ser Leu SerTyr His 50 55 60 7 59 PRT Bovine Xaa=unknown amino acid 7 Thr Leu LeuLys Tyr Glu Leu Arg Val Asp Leu Glu Asp Phe Glu Xaa 1 5 10 15 Asn ThrAla Phe Ala Lys Tyr Ala Asp Phe Ser Ile Ser Pro Asn Ala 20 25 30 Val SerAla Glu Glu Asp Gly Tyr Thr Leu Tyr Val Ser Gly Phe Glu 35 40 45 Asp GlyGly Ala Gly Asp Ser Leu Thr Tyr His 50 55 8 10 PRT Homo sapiens 8 LeuArg Val Glu Leu Glu Asp Ala Asn Ala 1 5 10 9 10 PRT Homo sapiens 9 TyrIle Val Lys Thr Ala Gly Asn Ala Leu 1 5 10 10 5 PRT Homo sapiens 10 GlnGlu Leu Glu Lys 1 5 11 11 PRT Homo sapiens 11 Phe Glu Asp Gly Val LeuAsp Pro Asp Tyr Pro 1 5 10 12 12 PRT Homo sapiens 12 Arg Phe Glu Asp GlyVal Leu Asp Pro Asp Tyr Pro 1 5 10 13 28 PRT Homo sapiens 13 Phe Cys LeuGln Gln Pro Leu Asp Cys Asp Asp Ile Tyr Ala Gln Gly 1 5 10 15 Tyr GlnSer Asp Gly Val Tyr Leu Ile Tyr Pro Ser 20 25 14 27 PRT bovine 14 SerGlu Leu Gln Leu Pro Leu Asp Glu Asp Asp Ile Tyr Ala Gln Gly 1 5 10 15Tyr Gln Ala Asp Gly Val Tyr Leu Ile Pro Ser 20 25 15 10 PRT Homo sapiens15 Ser Glu Leu Glu Lys His Gln Leu Asp Thr 1 5 10 16 374 PRT ArtificialSequence Description of Artificial Sequence Source Synthesized 16 MetAsp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15Leu Pro Gly Ala Arg Cys Asp Ile Gln Leu Thr Gln Ser Pro Ser Leu 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Met Ile Thr Cys Arg Ala Ser 35 40 45Gln Ala Ile Ser Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile His Ala Ala Ser Ser Leu Gln Thr Gly Val 65 70 7580 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Xaa Phe Thr Leu Thr 85 9095 Ile Ser Xaa Leu Gln Ser Glu Xaa Leu Gln Leu Tyr Tyr Cys Gln His 100105 110 Leu Lys Gly Tyr Pro Ile Thr Phe Arg Pro Arg Asp Thr Xaa Gly Xaa115 120 125 Xaa Xaa Asn Cys Xaa Cys Thr Ile Xaa Ser Ser Ser Ser Arg HisLeu 130 135 140 Xaa Asn Ile Glu Ile Trp Xaa Cys Leu Cys Cys Xaa Ala CysTyr Xaa 145 150 155 160 Ile Thr Ser Xaa Pro Lys Lys Ala Lys Phe His TrpLys Val Asp Asn 165 170 175 Pro Ser Asn Arg Val Thr Pro Gln Lys Asn PhePro Xaa Gln Lys Val 180 185 190 Phe Glu Asn Phe Gly Gln Gly Lys Xaa GlyXaa Lys Gly Xaa Gly Xaa 195 200 205 Xaa Xaa Xaa Phe Phe Phe Xaa Pro PheGly Xaa Xaa Xaa Xaa Phe Gly 210 215 220 Xaa Xaa Cys Xaa Cys Trp Xaa ProGly Xaa Xaa Lys Ile Phe Xaa Xaa 225 230 235 240 Pro Gly Gly Ala Lys ValGln Gly Glu Gly Gly Lys Xaa Leu Pro Ile 245 250 255 Gly Xaa Phe Pro XaaGlu Cys Xaa Gln Ser Xaa Thr Ala Arg Thr Ala 260 265 270 Leu Thr Ala SerAla Ala Pro Thr Arg Lys His Lys Val Tyr Ala Lys 275 280 285 Glu Val ThrHis Gln Gly Leu Pro Val Thr Lys Ser Xaa Asn Arg Gly 290 295 300 Glu CysXaa Xaa Arg Glu Lys Cys Pro His Leu Xaa Xaa Ser Ser Ser 305 310 315 320Leu Thr Pro Ser His Pro Leu Ala Xaa Xaa Asp Pro Phe Ser Thr Gly 325 330335 Asp Leu Pro Leu Leu Arg Ser Ser Ser Xaa Phe Phe Thr Ser Pro Pro 340345 350 Ser Ser Ser Leu Ala Xaa Ile Phe Ala Leu Ile Met Leu Met Leu Glu355 360 365 Glu Asn Glu Xaa Ile Lys 370 17 235 PRT Homo sapiens 17 MetAsp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15Leu Pro Gly Ala Arg Cys Ala Ile Arg Ile Ala Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser Thr Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile Ser Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile 65 70 7580 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 9095 Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Gly Gln Gln 100105 110 Tyr Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro SerAsp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Gly Leu LeuAsn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys ValAsp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr GluGln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu ThrLeu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Gly GluVal Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe AsnArg Gly Glu 225 230 235 18 28 PRT Artificial Sequence Description ofArtificial Sequence Source Synthesized 18 Leu Leu Leu Trp Ile Gly AlaAsp Ile Thr Gln Ser Pro Leu Val Ser 1 5 10 15 Gly Glu Arg Ala Thr IleAsn Cys Arg Ser Ser Gln 20 25 19 4 PRT Homo sapiens 19 Leu Leu Ala Leu 120 6 PRT Tetrahymena thermophila 20 Arg Ile Phe Glu Asn Tyr 1 5 21 5 PRTTetrahymena thermophila 21 Lys Gly Arg Lys Gly 1 5 22 4 PRT Homo sapiens22 Lys Leu Tyr Glu 1 23 4 PRT Homo sapiens 23 Ser Ser Ser Leu 1 24 8 PRTHomo sapiens 24 Ser Thr Gly Asp Ile Pro Met Leu 1 5 25 5 PRT Homosapiens 25 Thr Ser Thr Ala Asp 1 5 26 5 PRT cytomegalovirus 26 Phe PhePhe Ser Pro 1 5 27 4 PRT Homo sapiens 27 Phe Phe Phe Ser 1 28 4 PRT Homosapiens 28 Phe Phe Tyr Ser 1 29 328 PRT Artificial Sequence Descriptionof Artificial Sequence Source Synthesized 29 Met Val Leu Gln Thr Gln ValPhe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Asn Gly Asp IleVal Met Thr Gln Ser Pro Asp Ser Leu Gly 20 25 30 Val Ser Leu Gly Glu ArgAla Thr Ile Asn Cys Arg Ser Ser Gln Arg 35 40 45 Leu Phe Phe Gly Ser AsnSer Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60 Lys Pro Gly Gln Ser ProLys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65 70 75 80 Asp Ser Gly Val LeuThr Asp Ser Leu Ala Ala Gly Leu Gly Xaa Ile 85 90 95 Ser Leu Ser Pro SerXaa Xaa Cys Arg Leu Lys Asn Leu Ala Ile Leu 100 105 110 Xaa Leu Ser AlaIle Ile Ile Ile Ser Xaa Xaa Thr Phe Arg Pro Trp 115 120 125 Gly Thr XaaLeu Xaa Ile Gln Xaa Lys Cys Trp Xaa Ala Xaa Ile Phe 130 135 140 Xaa SerPhe Phe Pro Pro Xaa Glu Lys Gln Phe Lys Phe Phe Phe Phe 145 150 155 160Ser Pro Phe Leu Xaa Gly Trp Xaa Leu Gly Xaa Leu Phe Xaa Gly Pro 165 170175 Xaa Glu Lys Ile Phe Phe Pro Xaa Gly Pro Lys Lys Arg Gly Arg Gly 180185 190 Xaa Lys Xaa Pro Pro Asn Trp Gly Lys Ser Pro Ser Gly Xaa Xaa Xaa195 200 205 Gly Arg Gly Xaa Gln Gly Lys Gly Asn Leu Lys Ala Leu Trp XaaGlu 210 215 220 Pro Xaa Arg Leu Gly Lys Gly Gly Ile Arg Gly Xaa Asn LysXaa Xaa 225 230 235 240 Ala Xaa Glu Val Thr His Ser Gly Leu Ser Phe AlaXaa Ser Lys Lys 245 250 255 Xaa Xaa Gln Gly Arg Xaa Leu Glu Gly Glu ValPro Pro Pro Val Xaa 260 265 270 Xaa Xaa Gln Pro Asp Pro Leu Pro Ser PheGly Leu Xaa Pro Phe Phe 275 280 285 His Arg Gly Xaa Thr Pro Ile Xaa ValXaa Gln Xaa Ile Phe Tyr Xaa 290 295 300 Thr Pro Leu Xaa Xaa Leu Gly PheAsn Tyr Xaa Asn Val Xaa Xaa Xaa 305 310 315 320 Xaa Ile Asn Lys Val XaaPhe Leu 325 30 239 PRT Homo sapiens 30 Met Val Leu Gln Thr Gln Val PheIle Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Gly Asp Ile Val MetThr Gln Ser Pro Asp Ser Leu Ala Val 20 25 30 Ser Leu Gly Glu Arg Ala ThrIle Asn Cys Lys Ser Ser Gln Ser Val 35 40 45 Leu Tyr Ser Ser Asn Asn LysAsn Tyr Leu Ala Trp Tyr Gln Gln Lys 50 55 60 Pro Gly Gln Ala Pro Arg LeuLeu Ile Tyr Asp Ala Ser Ser Arg Ala 65 70 75 80 Thr Gly Ile Pro Asp ArgPhe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Thr Ile Ser ArgLeu Glu Pro Glu Asp Phe Ala Val Tyr Tyr 100 105 110 Gly Gln Gln Tyr GlySer Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys 115 120 125 Val Glu Ile LysArg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser AspGlu Gln Leu Lys Glu Ile Thr Ala Ser Val Val Gly Leu 145 150 155 160 LeuAsn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195200 205 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Gly Glu Val Thr His Gln210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Gly225 230 235 31 21 PRT Artificial Sequence Description of ArtificialSequence Source Synthesized 31 Phe Phe Phe Pro Phe Ala Ala Ala Lys LysVal Tyr Ala Glu Val Thr 1 5 10 15 His Gly Leu Ser Glu 20 32 4 PRT bovineand Homo sapiens 32 Ser Ile Ser Pro 1 33 5 PRT Homo sapiens 33 Leu LeuLeu Leu Ser 1 5 34 7 PRT cytomegalovirus 34 Cys Arg Ile Lys Asn Ala Val1 5 35 4 PRT Homo sapiens 35 Phe Tyr Tyr Ser 1 36 40 PRT ArtificialSequence Description of Artificial Sequence Source Synthesized 36 TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile His Ala 1 5 10 15Ala Ser Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 20 25 30Ser Gly Thr Xaa Phe Thr Leu Thr 35 40 37 40 PRT Artificial SequenceDescription of Artificial Sequence Source Synthesized 37 Trp Tyr Gln GlnLys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala 1 5 10 15 Ala Ser ThrLeu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 20 25 30 Ser Gly ThrAsp Phe Thr Leu Thr 35 40 38 40 PRT Homo sapiens 38 Ser Gly Pro Arg PheCys Gly Ser Val Ser Phe Phe Arg Gly Trp Asn 1 5 10 15 Asp Tyr Lys LeuGly Phe Gly Arg Ala Asp Gly Glu Tyr Trp Leu Gly 20 25 30 Leu Gln Asn MetHis Leu Leu Thr 35 40 39 40 PRT Bovine 39 Phe Gln Lys Arg Phe Asn GlySer Val Ser Phe Phe Arg Gly Trp Asn 1 5 10 15 Asp Tyr Lys Leu Gly PheGly Arg Ala Asp Gly Glu Tyr Trp Leu Gly 20 25 30 Leu Gln Asn Met His LeuLeu Thr 35 40 40 59 PRT sea cucumber 40 Phe Gln Arg Arg Ile Asp Gly ThrIle Asn Phe Tyr Arg Ser Trp Ser 1 5 10 15 Tyr Tyr Gln Thr Gly Phe GlyAsn Leu Asn Thr Glu Phe Trp Leu Gly 20 25 30 Asn Asp Asn Ile His Tyr LeuThr Phe Gln Arg Gly Phe Arg Trp Tyr 35 40 45 Gly Phe Gly Glu Trp Leu GlyAsn His Leu Thr 50 55 41 40 PRT Homo sapiens 41 Phe Gln Lys Arg Phe AsnGly Ser Val Ser Phe Phe Arg Gly Trp Asn 1 5 10 15 Asp Tyr Lys Leu GlyPhe Gly Arg Ala Asp Gly Glu Tyr Trp Leu Gly 20 25 30 Leu Gln Asn Met HisLeu Leu Thr 35 40 42 233 PRT Homo sapiens 42 Met Lys Lys Thr Ala Ile AlaIle Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala AlaGlu Leu Thr Gln Ser Pro Ser Ser Val Ser 20 25 30 Ala Ser Val Gly Asp ArgVal Thr Ile Thr Cys Arg Ala Ser Gln Gly 35 40 45 Ile Ser Ser Trp Leu AlaTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile Tyr SerAla Ser Ser Leu Gln Ser Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly SerGly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro GluAsp Ser Ala Thr Tyr Tyr Cys Gln Gln Ala Asn 100 105 110 Ser Phe Pro TyrThr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125 Thr Val AlaAla Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140 Leu LysSer Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His195 200 205 Lys Leu Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser ProVal 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 43 135 PRTHomo sapiens 43 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu TrpIle Ser 1 5 10 15 Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro AspSer Leu Ala 20 25 30 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys SerSer Gln Ser 35 40 45 Leu Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala TrpTyr Gln Gln 50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp AlaSer Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser GlySer Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu AspVal Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Pro MetPhe Gly Gln Gly Thr 115 120 125 Lys Val Glu Ile Lys Arg Thr 130 135 44127 PRT bovine Xaa=unknown amino acid 44 Ser Glu Leu Gln Leu Pro Leu AspGlu Asp Asp Ile Tyr Ala Gln Gly 1 5 10 15 Tyr Gln Ala Asp Gly Val TyrLeu Ile Pro Ser Gly Pro Arg Phe Cys 20 25 30 Gly Ser Val Ser Phe Phe ArgGly Trp Asn Asp Tyr Lys Leu Gly Phe 35 40 45 Gly Arg Ala Asp Gly Glu TyrTrp Leu Gly Leu Gln Asn Met His Leu 50 55 60 Leu Thr Leu Lys Tyr Glu LeuArg Val Asp Leu Glu Asp Phe Glu Xaa 65 70 75 80 Asn Thr Ala Phe Ala LysTyr Ala Asp Phe Ser Ile Ser Pro Asn Ala 85 90 95 Val Ser Ala Glu Glu AspGly Tyr Thr Leu Tyr Val Ser Gly Phe Glu 100 105 110 Asp Gly Gly Ala GlyAsp Ser Leu Thr Tyr His Ser Gly Gln Lys 115 120 125 45 252 PRT Homosapiens Xaa=unknown amino acid 45 Met Lys Ala Leu Leu Ala Leu Pro LeuLeu Leu Leu Leu Ser Thr Pro 1 5 10 15 Pro Cys Ala Pro Gln Val Ser GlyIle Arg Gly Asp Ala Leu Glu Arg 20 25 30 Phe Cys Leu Gln Gln Pro Leu AspCys Asp Asp Ile Tyr Ala Gln Gly 35 40 45 Tyr Gln Ser Asp Gly Val Tyr LeuIle Tyr Pro Ser Gly Pro Ser Val 50 55 60 Pro Val Pro Val Phe Cys Asp MetThr Thr Glu Gly Gly Lys Trp Thr 65 70 75 80 Val Phe Gln Lys Arg Phe AsnGly Ser Val Ser Phe Phe Arg Gly Trp 85 90 95 Asn Asp Tyr Lys Leu Gly PheGly Arg Ala Asp Gly Glu Tyr Trp Leu 100 105 110 Gly Leu Gln Asn Met HisLeu Leu Thr Leu Lys Gln Lys Tyr Glu Leu 115 120 125 Arg Val Asp Leu GluAsp Phe Glu Asn Asn Thr Ala Tyr Ala Lys Tyr 130 135 140 Ala Asp Phe SerIle Ser Pro Asn Ala Val Ser Ala Glu Glu Asp Gly 145 150 155 160 Xaa ThrLeu Phe Val Ala Gly Phe Glu Asp Gly Gly Ala Gly Asp Ser 165 170 175 LeuSer Tyr His Ser Gly Gln Lys Phe Ser Thr Phe Asp Arg Asp Gln 180 185 190Asp Leu Phe Val Gln Asn Cys Ala Ala Leu Ser Ser Gly Ala Arg Trp 195 200205 Phe Arg Ser Cys His Phe Ala Asn Leu Asn Gly Phe Tyr Leu Gly Gly 210215 220 Ser Leu Ser Tyr Ala Asn Gly Ile Asn Trp Trp Lys Gly Phe Tyr Tyr225 230 235 240 Ser Leu Lys Arg Thr Glu Met Lys Ile Arg Arg Ala 245 25046 282 PRT sea cucumber 46 Met Phe Ser Phe Ile Met Lys Ala Ala Ile LeuLeu Ile Leu Val Gly 1 5 10 15 Cys Ile Ser Phe Cys Ile Ser Ser Glu ProLeu Asn Glu Ser Glu Ile 20 25 30 Thr Phe Glu Arg Glu Glu Arg Ser Leu AlaAsp Pro Ala Gly Arg Gln 35 40 45 Lys Arg Gln Ser Gly Leu Ser Cys Pro LysArg Ile Ser His Ser Pro 50 55 60 Glu Tyr Pro Arg Asp Cys Tyr Asp Ile LeuGln Ser Cys Ser Gly Gln 65 70 75 80 Ser Pro Pro Ser Gly Gln Tyr Tyr IleGln Pro Asp Gly Gly Asn Leu 85 90 95 Ile Lys Val Tyr Cys Asp Met Glu ThrAsp Glu Gly Gly Trp Thr Val 100 105 110 Phe Gln Arg Arg Ile Asp Gly ThrIle Asn Phe Tyr Arg Ser Trp Ser 115 120 125 Tyr Tyr Gln Thr Gly Phe GlyAsn Leu Asn Thr Glu Phe Trp Leu Gly 130 135 140 Asn Asp Asn Ile His TyrLeu Thr Ser Gln Gly Asp Tyr Glu Leu Arg 145 150 155 160 Val Glu Leu AsnAsn Thr Leu Gly Asn His Tyr Tyr Ala Lys Tyr Asn 165 170 175 Lys Phe ArgIle Gly Asp Ser Phe Ser Glu Tyr Leu Leu Val Leu Gly 180 185 190 Ala TyrSer Gly Thr Ala Gly Asp Ser Leu Ala Tyr His Asn Thr Met 195 200 205 ArgPhe Ser Thr Tyr Asp Asn Asp Asn Asp Val Tyr Ser Ile Asn Cys 210 215 220Ala Ser His Ser Ser Tyr Gly Arg Gly Ala Trp Trp Tyr Lys Ser Cys 225 230235 240 Leu Leu Ser Asn Leu Asn Gly Gln Tyr Tyr Asp Tyr Ser Gly Ala Pro245 250 255 Ser Ile Tyr Trp Ser Tyr Leu Pro Gly Asp Asn Asp Gln Ile ProPhe 260 265 270 Ala Glu Met Lys Leu Arg Asn Arg Ser Ile 275 280 47 11PRT Homo sapiens 47 Phe Glu Asp Gly Val Leu Asp Pro Asp Tyr Pro 1 5 1048 12 PRT Homo sapiens 48 Arg Phe Glu Asp Gly Val Leu Asp Pro Asp TyrPro 1 5 10 49 6 PRT homo sapiens and bovine 49 Asp Gly Val Tyr Leu Pro 15 50 7 PRT Homo sapiens 50 Tyr Glu Leu Arg Ser Leu Ser 1 5 51 4 PRT Homosapiens 51 Tyr Glu Leu Lys 1 52 4 PRT cytomegalovirus 52 Ser Leu Ser Ala1 53 5 PRT Homo sapiens 53 Arg Gly Phe Tyr Tyr 1 5 54 5 PRT Homo sapiens54 Arg Val Tyr Phe Phe 1 5 55 4 PRT cytomegalovirus 55 Glu Glu Lys Asn 156 4 PRT Homo sapiens 56 Tyr Gly Leu Arg 1 57 4 PRT Homo sapiens 57 TrpGly Phe Thr 1

What is claimed is:
 1. A purified protein comprising the amino acidsequence shown in SEQ ID NO:2.
 2. A purified protein comprising theamino acid sequence shown in SEQ ID NO:3.
 3. A purified proteincomprising the amino acid sequence shown in SEQ ID NO:4.
 4. A kit fordetecting in a sample the presence of an immunoglobulin which can bindto the protein of claim 1, 2 or 3, which comprises: (a) a solid supporthaving probes linked thereto, which probes may be the same or different,each probe comprising the protein of claim 1, 2 or 3; and (b) a meansfor determining the presence of immunoglobulin bound to the probes. 5.The kit of claim 4, wherein the means for determining the presence ofthe immunoglobulin is a detectable label.
 6. The kit of claim 5, whereinthe detectable label is a radioactive isotope, an enzyme, a dye, biotin,a flourescent label or a chemiluminescent label.
 7. The protein of claim1, 2 or 3, wherein the protein binds to a human kappa immunoglobulin. 8.The protein of claim 1, 2 or 3, wherein the protein is a recombinantprotein.